U.S. patent application number 15/756947 was filed with the patent office on 2018-10-18 for compounds and compositions for targeting brain injuries and methods of use thereof.
This patent application is currently assigned to SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE. The applicant listed for this patent is Sazid HUSSAIN, Aman MANN, Erkki RUOSLAHTI, SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE, Pablo SCODELLER. Invention is credited to Sazid HUSSAIN, Aman MANN, Erkki RUOSLAHTI, Pablo SCODELLER.
Application Number | 20180296696 15/756947 |
Document ID | / |
Family ID | 56943950 |
Filed Date | 2018-10-18 |
United States Patent
Application |
20180296696 |
Kind Code |
A1 |
RUOSLAHTI; Erkki ; et
al. |
October 18, 2018 |
COMPOUNDS AND COMPOSITIONS FOR TARGETING BRAIN INJURIES AND METHODS
OF USE THEREOF
Abstract
Disclosed are methods and compositions for selectively targeting
sites of traumatic brain injury (TBI). A brain injury-specific
4-amino acid peptide (sequence CAQK), identified by in vivo phage
display screening in mice with acute brain injury, shows selective
binding to mouse and human brain injury lesions, and when
systemically injected, specifically homes to sites of injury in
penetrating and non-penetrating (controlled cortical impact) brain
injury models. Also disclosed are methods and compositions for
delivering therapeutic compounds to such sites. CAQK-coated
nanoparticles containing silencing oligonucleotides provide an
alternative to local delivery of therapeutics, which is invasive
and can add complications to the injury.
Inventors: |
RUOSLAHTI; Erkki; (La Jolla,
CA) ; MANN; Aman; (La Jolla, CA) ; SCODELLER;
Pablo; (La Jolla, CA) ; HUSSAIN; Sazid; (La
Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RUOSLAHTI; Erkki
MANN; Aman
SCODELLER; Pablo
HUSSAIN; Sazid
SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE |
La Jolla
La Jolla
La Jolla
La Jolla
La Jolla |
CA
CA
CA
CA
CA |
US
US
US
US
US |
|
|
Assignee: |
SANFORD BURNHAM PREBYS MEDICAL
DISCOVERY INSTITUTE
La Jolla
CA
|
Family ID: |
56943950 |
Appl. No.: |
15/756947 |
Filed: |
September 2, 2016 |
PCT Filed: |
September 2, 2016 |
PCT NO: |
PCT/US16/50168 |
371 Date: |
March 1, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62213168 |
Sep 2, 2015 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 48/005 20130101;
A61K 47/6923 20170801; A61K 47/6425 20170801; A61K 9/0019 20130101;
A61K 47/6929 20170801; A61K 47/62 20170801; A61K 48/0008 20130101;
A61K 9/5115 20130101; C07K 5/1013 20130101; A61P 43/00
20180101 |
International
Class: |
A61K 47/69 20060101
A61K047/69; A61K 9/51 20060101 A61K009/51; A61K 9/00 20060101
A61K009/00; A61P 43/00 20060101 A61P043/00; A61K 48/00 20060101
A61K048/00; A61K 47/64 20060101 A61K047/64 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under
Cooperative Agreement HR0011-13-2-0017 awarded by the Defense
Advanced Research Projects Agency (DARPA). The Government has
certain rights in the invention.
Claims
1-24. (canceled)
25. A method of selectively targeting a cargo composition to a site
of nervous system injury in a subject, the method comprising:
administering a composition comprising an isolated peptide
comprising the amino acid sequence CAQK (SEQ ID NO:4) to a subject
having a nervous system injury, wherein the composition selectively
homes to a site of the nervous system injury in the subject thereby
selectively targeting the cargo composition of the composition to
the site of the nervous system injury.
26. The method of claim 25, wherein the nervous system injury
comprises a brain injury, wherein the peptide selectively homes to
a site of the brain injury.
27. The method of claim 26, wherein the brain injury comprises
traumatic brain injury, stroke injury, or both.
28. The method of claim 25, wherein the nervous system injury is a
central nervous system injury, a peripheral nervous system injury,
a brain injury, a spinal cord injury, a neural injury, a neuronal
injury, or combinations thereof.
29. The method of claim 25, wherein the nervous system injury is an
autoimmune disease that affects nerves or parts or components of
the nervous system, a demyelinating disease, multiple sclerosis, or
combinations thereof.
30. The method of claim 25, wherein the nervous system injury is
acute.
31. The method of claim 25, wherein the nervous system injury is
chronic.
32. The method of claim 25, wherein the peptide specifically binds
to one or more of versican, tenascin-R, and Hapln.
33. The method of claim 25, wherein the peptide selectively homes
to a site of glial scar formation.
34. The method of claim 25, wherein the peptide selectively homes
to a site where hyaluronic acid, versican, tenascin-R, and Hapln
are being deposited.
35. The method of claim 25, wherein the composition is administered
intravenously.
36. The method of claim 25, wherein the composition is administered
systemically.
37. The method of claim 25, wherein the composition is administered
within 10 days of the onset of the nervous system injury.
38. The method of claim 25, wherein the composition is administered
within 5 days of the onset of the nervous system injury.
39. The method of claim 25, wherein the composition is administered
within 24 hours of the onset of the nervous system injury.
40. A method of selectively targeting a cargo composition to a site
of nervous system injury in a subject, the method comprising:
administering a composition to a subject having a nervous system
injury, wherein the composition comprises a peptide and a cargo
composition, wherein the peptide consists of the amino acid
sequence CAQK (SEQ ID NO:4), wherein the cargo composition
comprises a surface molecule and cargo molecule, wherein the
surface molecule comprises a nanoparticle, wherein the cargo
molecule is encapsulated in the nanoparticle, wherein the cargo
molecule is a therapeutic agent, and wherein the therapeutic agent
comprises a functional nucleic acid, wherein the composition
selectively homes to a site of the nervous system injury in the
subject thereby selectively targeting the cargo composition of the
composition to the site of the nervous system injury.
41-59. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 62/213,168, filed Sep. 2, 2015. Application No.
62/213,168, filed Sep. 2, 2015, is hereby incorporated herein by
reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0003] The Sequence Listing submitted on Sep. 2, 2016 as a text
file named "SBMRI_16-002_PCT_ST25", created on Aug. 19, 2016, and
having a size of 2,717 bytes is hereby incorporated by
reference.
FIELD OF THE INVENTION
[0004] The disclosed invention relates generally to the fields of
molecular medicine and brain biology and, more specifically, to
molecules that selectively home to sites of acute brain injury.
BACKGROUND OF THE INVENTION
[0005] Acute brain injury such as traumatic brain injury (TBI)
disrupts the normal function of the brain and generally has a poor
prognosis for functional recovery and survival. Termed a `silent
epidemic`, TBI is a leading cause of mortality and morbidity in
children, teens and active adults from ages 1 to 44, with an annual
incidence of 2.5 million in the US (Coronado et al., J. Safety Res.
43:299-307 (2012)). TBI can lead to acute and potentially
long-lasting neurological dysfunction, including the development of
chronic traumatic encephalopathy (CTE) or even Alzheimer's disease
(Smith et al., Nat. Rev. Neurology 9:211-221 (2013)). A majority of
combat-related TBI cases are additionally complicated by a
penetrating injury to the brain, which is often even more difficult
to manage than non-penetrating injuries (Bell et al., J. Trauma
66:S104-111 (2009)). Despite this substantial socio-economic
impact, TBI treatment is limited to palliative care and no specific
therapies with long-term benefits are available.
[0006] The blood-brain barrier (BBB) is considered a major
impediment to systemic treatment of central nervous system (CNS)
diseases. As a result, localized delivery of drugs within the brain
has been explored, but it has limitations in clinical settings. In
acute brain injury and several cerebrovascular diseases, including
stroke, hypertension, and ischemia, the BBB is transiently
disrupted, which allows extravascular access for macromolecules and
neuroprotective drugs from the systemic circulation. In fact, the
leakage of serum proteins into brain parenchyma is used to test for
BBB integrity (Kuroiwa et al., Acta Neuropathologica 76:62-70
(1988)). However, lack of specific binding of passively
accumulating proteins in the injured area can result in low
retention and subsequent washout over time. Due to this clearance,
the therapeutic efficacy of a systemically administered drug may be
greatly limited.
[0007] Previous studies have used in vivo phage display to probe
tissues in situ for specific molecular signatures and discovered
homing peptides specific for different pathologies including
tumors, atherosclerotic plaques, and wounds (Ruoslahti, Nat. Rev.
Cancer 2:83-90 (2002); Ruoslahti, Adv. Mater. 24:3747-3756 (2012);
Teesalu et al., Methods Enzymol. 503:35-56 (2012)). An acute and
complex event such as TBI is suited for a similar approach as
site-specific molecular changes in protein expression have been
reported (Natale et al., J. Neurotrauma 20:907-927 (2003)).
[0008] Current approaches for delivering therapeutics to brain
injury sites are invasive and can add complications to the
injury.
[0009] It is an object of the present invention to provide peptides
that recognize specific molecular changes at the sites of nervous
system injury and enhance delivery of compounds and compositions to
such sites.
[0010] It is another object of the present invention to provide
peptides that selectively home to sites of nervous system
injury.
[0011] It is another object of the present invention to provide
compositions that selectively home to sites of nervous system
injury.
[0012] It is another object of the present invention to provide
methods for selectively targeting sites of nervous system
injury.
[0013] It is another object of the present invention to provide
methods for treating nervous system injury by selectively targeting
sites of nervous system injury.
[0014] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is not to be taken as an admission that any or all of
these matters form part of the prior art base or were common
general knowledge in the field relevant to the present disclosure
as it existed before the priority date of each claim of this
application.
[0015] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
BRIEF SUMMARY OF THE INVENTION
[0016] The methods and compositions provided herein are based on
the finding that a 4-amino acid peptide (CAQK) selectively
recognizes brain injuries and accumulates at the sites of injury.
This and other peptides containing the CAQK amino acid sequence
were discovered to enhance the accumulation of systemically
administered payloads with chemistries ranging in size from a
drug-sized molecule to nanoparticles, and incorporating a variety
of imaging and therapeutic functions of potential utility in
clinical management of brain injuries. Importantly, the target of
this delivery system is expressed both in mouse and human brain
injuries.
[0017] The binding target for the CAQK peptide is a chondroitin
sulfate proteoglycan (CSPG)-rich protein-carbohydrate extracellular
matrix complex that is overexpressed in CNS injuries and is notably
composed of hyaluronic acid, versican, aggrecan, brevican,
neurocan, phosphacan, tenascin-R, and hyaluronan and proteoglycan
link protein (Hapln). The overexpression of this complex results in
the formation of a CSPG-rich glial scar, which is a major barrier
to regeneration. This CSPG-rich extracellular matrix complex is
also overexpressed in stroke and other nervous system injuries.
Degradation of the complex is known to improve the outcome of
stroke. CAQK-containing peptides were also shown to the site of
injury in an experimental model of stroke caused by cerebral
ischemia. Thus, the discovered peptides can selectively target
sites of nervous system injury, especially those that overexpress
CSPGs.
[0018] Disclosed are peptides, compositions, and methods for
selective targeting nervous system injury, such as brain injury and
stroke injury, sites of glial scar formation, and sites where
hyaluronic acid, versican, tenascin-R, and Hapln are being
deposited. The disclosed peptides, compositions, and methods are
useful for selectively targeting acute nervous system injury, such
as traumatic brain injury and stroke injury.
[0019] A peptide sequence that specifically homes to sites of
nervous system injury has been discovered. Peptides containing the
sequence home to, and can delivery large cargos to, sites of
nervous system injury in the brain without regard to the blood
brain barrier. This is likely due to compromise of the blood brain
barrier in nervous system injury. The targeting and selectively
homing are due to the presence of molecules at the site of nervous
system injury to which the peptide sequence can bind.
[0020] Disclosed are peptides that contain the amino acid sequence
CAQK (SEQ ID NO:4). In some forms, the peptides have any one or
combinations of the following properties: selective homing to a
site of nervous system injury; selective homing to a site of acute
nervous system injury; selective homing to a site of brain injury;
selective homing to a site of acute brain injury; selective homing
to a site of stroke injury; selective homing to a site of acute
stroke injury; specific binding to one or more of versican,
tenascin-R, and Hapln; selective homing to a site of glial scar
formation; selective homing to a site where hyaluronic acid,
versican, tenascin-R, and Hapln are being deposited; and selective
homing to CSPG-rich extracellular matrix complex.
[0021] In some forms, the peptide is 100 amino acids in length or
less, 50 amino acids in length or less, 30 amino acids in length or
less, 20 amino acids in length or less, 15 amino acids in length or
less, 10 amino acids in length or less, 8 amino acids in length or
less, 6 amino acids in length or less, 5 amino acids in length or
less, or 4 amino acids in length. In some forms, the peptide is
linear. In some forms, the peptide is circular.
[0022] In some forms of the peptide, the amino acid sequence CAQK
(SEQ ID NO:4) is at the C terminal end of the peptide. In some
forms, the peptide consists of the amino acid sequence CAQK (SEQ ID
NO:4). In some forms, the peptide is modified. In some forms, the
peptide is a methylated peptide. In some forms, the peptide
includes a methylated amino acid segment. In some forms, the
peptide is N- or C-methylated in at least one position.
[0023] Disclosed are compositions that include the disclosed
peptide. In some forms, the composition includes the disclosed
peptide and a cargo composition. In some forms of the composition,
the peptide and the cargo composition are covalently coupled or
non-covalently associated with each other. In some forms, the
composition includes the disclosed peptide and a cargo molecule. In
some forms of the composition, the peptide and the cargo molecule
are covalently coupled or non-covalently associated with each
other.
[0024] In some forms, the composition selectively homes to a site
of acute brain injury. In some forms, the composition specifically
binds to one or more of versican, tenascin-R, and Hapln. In some
forms, the composition selectively homes to a site of glial scar
formation. In some forms, the composition selectively homes to a
site where hyaluronic acid, versican, tenascin-R, and Hapln are
being deposited. In some forms, the composition selectively homes
to CSPG-rich extracellular matrix complex. In some forms, the
composition selectively homes to a site of acute brain injury,
specifically binds to one or more of versican, tenascin-R, and
Hapln, selectively homes to a site of glial scar formation,
selectively homes to a site where hyaluronic acid, versican,
tenascin-R, and Hapln are being deposited, selectively homes to
CSPG-rich extracellular matrix complex, or combinations
thereof.
[0025] In some forms, the cargo composition includes one or more
cargo molecules. In some forms of the cargo composition, the cargo
molecules each independently include a therapeutic agent, a
therapeutic protein, a therapeutic compound, a therapeutic
composition, a polypeptide, a nucleic acid molecule, a small
molecule, a label, a labeling agent, a contrast agent, an imaging
agent, a fluorophore, fluorescein, rhodamine, a radionuclide,
indium-111, technetium-99, carbon-11, or carbon-13, or combinations
thereof. In some forms of the cargo composition, at least one of
the cargo molecules includes a therapeutic agent. In some forms of
the cargo composition, at least one of the cargo molecules includes
a functional nucleic acid. In some forms of the cargo composition,
at least one of the cargo molecules includes a detectable
agent.
[0026] In some forms, the cargo composition includes a surface
molecule. In some forms, the cargo composition further includes a
surface molecule. In some forms, the surface molecule includes a
nanoparticle, a nanoworm, an iron oxide nanoworm, an iron oxide
nanoparticle, an albumin nanoparticle, a liposome, a micelle, a
phospholipid, a polymer, a microparticle, a bead, a virus, a phage,
a viral particle, a phage particle, a viral capsid, a phage capsid,
a virus-like particle, or a fluorocarbon microbubble. In some
forms, the surface molecule includes a nanoparticle. In some forms
of the composition, the cargo molecules are encapsulated in the
nanoparticle.
[0027] In some forms, the composition includes a plurality of the
cargo composition. In some forms, the composition includes a
plurality of the peptide. In some forms of the composition, the
peptide and the cargo composition are coupled via a linker.
[0028] Disclosed are compositions comprising a peptide and a cargo
composition, where the peptide consists of the amino acid sequence
CAQK (SEQ ID NO:4), where the cargo composition includes a surface
molecule and cargo molecule, where the surface molecule includes a
nanoparticle, where the cargo molecule is encapsulated in the
nanoparticle, where the cargo molecule is a therapeutic agent, and
where the therapeutic agent includes a functional nucleic acid.
[0029] The disclosed peptides and compositions are useful for
selective targeting nervous system injury, such as brain injury and
stroke injury, sites of glial scar formation, sites where
hyaluronic acid, versican, tenascin-R, and Hapln are being
deposited, and CSPG-rich extracellular matrix complexes. For
example, the disclosed peptides and compositions are useful for
selectively targeting acute nervous system injury, such as
traumatic brain injury and stroke injury. Thus, methods for
selectively targeting a cargo to a site of acute nervous system
injury in a subject are disclosed.
[0030] In some forms, the method involves administering the
disclosed composition to a subject having an acute nervous system
injury. The composition selectively homes to a site of the nervous
system injury in the subject thereby selectively targeting the
cargo composition of the composition to the site of the nervous
system injury.
[0031] In some forms, the nervous system injury includes a brain
injury. In some forms, the peptide selectively homes to a site of
the brain injury. In some forms, the brain injury includes
traumatic brain injury, stroke injury, or both. In some forms, the
peptide specifically binds to one or more of versican, tenascin-R,
and Hapln. In some forms, the peptide selectively homes to a site
of glial scar formation. In some forms, the peptide selectively
homes to a site where hyaluronic acid, versican, tenascin-R, and
Hapln are being deposited. In some forms, the peptide selectively
homes to CSPG-rich extracellular matrix complex.
[0032] The composition can be administered by any suitable route.
In some forms, the composition is administered intravenously. In
some forms, the composition is administered systemically. In some
forms, the composition is not administered locally.
[0033] The disclosed peptides and compositions are particularly
useful for targeting acute nervous system injury. Thus, in some
forms, the composition can be administered near the time of the
injury or during the acute phase of the injury. In some forms, the
composition is administered within 10 days of the onset of the
nervous system injury. In some forms, the composition is
administered within 5 days of the onset of the nervous system
injury. In some forms, the composition is administered within 24
hours of the onset of the nervous system injury.
[0034] Disclosed in particular are methods of selectively targeting
a cargo composition to a site of acute nervous system injury in a
subject, where the method involves administering the composition to
a subject having an acute nervous system injury, where the
composition includes a peptide and a cargo composition, where the
peptide consists of the amino acid sequence CAQK (SEQ ID NO:4),
where the cargo composition includes a surface molecule and cargo
molecule, where the surface molecule includes a nanoparticle, where
the cargo molecule is encapsulated in the nanoparticle, where the
cargo molecule is a therapeutic agent, and where the therapeutic
agent includes a functional nucleic acid. The composition
selectively homes to a site of the nervous system injury in the
subject thereby selectively targeting the cargo composition of the
composition to the site of the nervous system injury.
[0035] Also disclosed are methods of selectively targeting
CSPG-rich extracellular matrix complexes (extracellular matrix
containing hyaluronic acid, versican, tenascin-R, and Hapln and
exemplified by the matrix of cultured U251 astrocytoma cells). The
method can involve administering a composition to a subject, where
the composition comprises a homing molecule and a pharmaceutically
acceptable carrier. The composition selectively homes to the
CSPG-rich extracellular matrix complex thereby selectively
targeting the CSPG-rich extracellular matrix complex.
[0036] In some forms of the method, the homing molecule can
specifically bind to one or more of versican, tenascin-R, and
Hapln. In some forms of the method, the homing molecule can
selectively home to a site where hyaluronic acid, versican,
tenascin-R, and Hapln are being deposited. In some forms of the
method, the homing molecule can selectively home to CSPG-rich
extracellular matrix complex. In some forms of the method, the
composition can be administered intravenously. In some forms of the
method, the composition can be administered systemically.
[0037] In some forms of the method, the composition can further
comprise a cargo composition. In some forms of the method, the
cargo composition can include one or more cargo molecules. In some
forms of the method, the cargo molecules can each independently be
a therapeutic agent, a therapeutic protein, a therapeutic compound,
a therapeutic composition, a polypeptide, a nucleic acid molecule,
a small molecule, a label, a labeling agent, a contrast agent, an
imaging agent, a fluorophore, fluorescein, rhodamine, a
radionuclide, indium-111, technetium-99, carbon-11, or carbon-13,
or combinations thereof. In some forms of the method, at least one
of the cargo molecules comprises a therapeutic agent. In some forms
of the method, at least one of the cargo molecules comprises a
functional nucleic acid. In some forms of the method, at least one
of the cargo molecules comprises a detectable agent.
[0038] Also disclosed are homing molecules that selectively home to
CSPG-rich extracellular matrix complexes. In some forms, the homing
molecules can be identified by bringing into contact the homing
molecule and versican, tenascin-R, Hapln, or combinations thereof,
and assessing whether the homing molecule specifically binds to the
versican, tenascin-R, Hapln, or combination thereof. The homing
molecule is identified if the homing molecule specifically binds to
the versican, tenascin-R, Hapln, or combination thereof. In some
forms, the homing molecules can be identified by bringing into
contact the homing molecule and CSPG-rich extracellular matrix
complexes, and assessing whether the homing molecule specifically
binds to the CSPG-rich extracellular matrix complexes. The homing
molecule is identified if the homing molecule specifically binds to
the CSPG-rich extracellular matrix complexes. In some forms the
CSPG-rich extracellular matrix complexes can be matrix produced by
U251 astrocytoma cells.
[0039] Also disclosed are methods of identifying compounds that
target sites of nervous system injury. The method can involve
bringing into contact a test compound and versican, tenascin-R,
Hapln, or combinations thereof, and assessing whether the test
compound specifically binds to the versican, tenascin-R, Hapln, or
combination thereof. The test compound is identified as a compound
that target sites of nervous system injury if the test compound
specifically binds to the versican, tenascin-R, Hapln, or
combination thereof.
[0040] In some forms of the method, the versican, tenascin-R,
Hapln, or combination thereof can be part of extracellular matrix.
In some forms of the method, the extracellular matrix can be in or
obtained from glial scar. In some forms, the extracellular matrix
is CSPG-rich extracellular matrix complex (extracellular matrix
containing hyaluronic acid, versican, tenascin-R, and Hapln and
exemplified by the matrix of cultured U251 astrocytoma cells).
[0041] In some forms of the method, assessing whether the test
compound specifically binds the versican, tenascin-R, Hapln, or
combination thereof, can be accomplished by assessing whether the
test compound specifically binds the CSPG-rich extracellular matrix
complex. In some forms of the method, the extracellular matrix can
be at a site of nervous system injury in test animal, where
bringing into contact is accomplished by administering the test
compound to the animal intravenously.
[0042] In some forms of the method, the versican, tenascin-R,
Hapln, or combination thereof is not comprised in extracellular
matrix. In some forms of the method, the versican, tenascin-R,
Hapln, or combination thereof can be made from individual versican,
tenascin-R, and Hapln proteins.
[0043] In some forms of the method, the test compound can be
coupled to a label, wherein assessing whether the test compound
specifically binds is accomplished by detecting the label.
[0044] Additional advantages of the disclosed method and
compositions will be set forth in part in the description which
follows, and in part will be understood from the description, or
may be learned by practice of the disclosed method and
compositions. The advantages of the disclosed method and
compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosed method and compositions and together
with the description, serve to explain the principles of the
disclosed method and compositions.
[0046] FIG. 1A illustrates a schematic of the PBI mouse model where
a 5 mm craniotomy was performed in the right parietotemporal cortex
and nine needle punctures were inflicted according to the grid
shown. FIG. 1B is a graph showing CAQK phage frequency in brain as
percentage of total phage recovered. Compared to PBI, CAQK was
present at a lower percentage in the contralateral hemisphere in
injured mice, and absent in healthy, control mice. FIG. 1C is a
graph quantifying fluorescence brain images of mice injected with
FAM-labeled peptides six hours after PBI. Brains were perfused,
isolated, and imaged under an Illumatool System (green channel).
(*P<0.05, ANOVA analysis, n=6); Mean.+-.SEM. FIG. 1D is a graph
illustrating selective homing of a synthetic FAM-CAQK peptide to
the injured brain. The graph was compiled by quantifying and
plotting signal from immunohistochemical staining for FAM. FIG. 1E
is a graph illustrating a time course of CAQK accumulation in PBI.
FIG. 1F is a graph illustrating blood brain barrier leakage in PBI.
Brains isolated from mice after different time points after PBI
were stained for mouse IgG. Total fluorescence intensity was
quantified and plotted. Mean.+-.SEM, n=3.
[0047] FIG. 2 is a graph showing protein expression of PNN
components in PBI brain. Frozen sections of perfused mouse brains
six hours after PBI were immunostained and analyzed by confocal
microscopy. The graph shows quantification of immunofluorescence of
versican, Hapln4 and tenascin R in injured and contralateral
hemispheres in PBI brain. Signal intensity was quantified by taking
the integrated pixel intensity in the red channel of three images.
Mean.+-.SEM.
[0048] FIGS. 3A-3C are graphs illustrating colocalization of CAQK
with chondroitin sulfate in PBI. FIG. 3A illustrates phage binding
to extracellular matrix formed by U251 cells. The cells were gently
dissociated and removed, and the remaining ECM was incubated with
phage for 1 hour at room temperature, and phage binding was
detected following an ELISA protocol. CAQK phage showed higher
binding to ECM than control phage. FIG. 3B shows inhibition of CAQK
phage binding to ECM by free CAQK peptide. FIG. 3C illustrates that
phage binding to ECM is reduced upon enzymatic digestion of ECM
with chondroitinase ABC or hyaluronidase. Mean.+-.SEM.
Representative data shown in FIGS. 3A, 3B, and 3C is from three
experimental repetitions each with three sample replicates. FIG. 3D
is a graph showing versican expression in U251 cells. U251 cells
grown as confluent monolayer were stained for versican expression
and analyzed by immunofluorescence after no treatment or treatment
with hyaluronidase (50 U/ml) or chondroitinase ABC (3 U/ml) for 1
hour at 37.degree. C. Cells were counterstained with DAPI. Versican
was quantified and plotted in the bar graph. Representative of
three experiments is shown.
[0049] FIG. 4A is a graph illustrating CAQK-mediated delivery of
silver nanoparticles to PBI. Silver nanoparticles conjugated with
CAQK (CAQK-NPs) or a control peptide (control-NPs) were injected
i.v. 6 hours after PBI and allowed to circulate for two hours
before perfusion (n=3). FIGS. 4B-4D are graphs showing
characterization of PSiNPs. FIGS. 4B and 4C characterize CAQK
peptide-conjugated porous silicon nanoparticles (CAQK-PSiNPs) using
size distribution measured by dynamic light scattering (FIG. 4B);
and photoluminescence spectra (.lamda..sub.ex =365 nm) of PSiNP and
CAQK-PSiNP (FIG. 4C; CAQK-PSiNP shows peak at .about.530 nm while
PSiNP does not). FAM label on the peptide appeared in the emission
spectrum at .about.530 nm and was included to allow estimation of
conjugation efficiency to PSiNP, as described in the methods
section. FIG. 4D shows release of siRNA from PSiNPs at different
time points. Dy677-labeled siRNA was loaded into PSiNPs and
incubated in PBS at 37.degree. C. The released siRNA was obtained
from supernatant at different time points.
[0050] FIGS. 5A-5C illustrate siRNA delivery in PBI with
CAQK-conjugated PSiNPs. FIG. 5A is a schematic of the experimental
design for siRNA delivery. FIG. 5B is a graph showing
signal-to-noise ratio (SNR) calculated for the peptide-conjugated
PSiNPs in each mouse tissue (relative to PBS control) as described
in the methods. CAQK-PSiNP showed significantly higher SNR in PBI
brains than the control peptide-conjugated group (Mean.+-.SD,
*P<0.05, two-tailed Student's t test; n.s.--not significant,
n=3). FIG. 5C is a graph showing percentage GFP/DAPI expression.
Mean GFP intensity in injured hemisphere was normalized to
corresponding contralateral hemisphere and plotted as percentage
GFP expression (y-axis) (*P<0.05; ANOVA analysis, n=3). DAPI
signal (plotted) showed similar total cell number. Mean.+-.SEM,
n.s., not significant.
[0051] FIGS. 6A-6E are graphs illustrating CAQK binding to injured
human brains. FIGS. 6A and 6B show binding of CAQK-NPs to sections
of the corpus callosum (FIG. 6A) and cortex (FIG. 6B) of human
brains. Peptide-conjugated nanoparticles were incubated with
formalin fixed frozen sections from injured and normal brains for
ex vivo binding. Sections were counterstained with nuclear fast
red, and the number of particles in each frame were counted and
plotted in the graph. FIG. 6C illustrates versican expression in
human brains. FIGS. 6D and 6E illustrate Hapln4 expression in the
corpus callosum (FIG. 6D) and cortex (FIG. 6E) of human brains.
Positive immunohistochemical staining was quantified and plotted.
Mean.+-.SEM, (ANOVA analysis; *P<0.05, **P<0.005,
***P<0.0005).
DETAILED DESCRIPTION OF THE INVENTION
[0052] The disclosed method and compositions may be understood more
readily by reference to the following detailed description of
particular embodiments and the Example included therein and to the
Figures and their previous and following description.
[0053] A 4-amino acid peptide (CAQK) was discovered that
selectively recognizes brain injuries and accumulates at the sites
of injury. Peptides containing this sequence enhance the
accumulation of systemically administered payloads with chemistries
ranging in size from a drug-sized molecule to nanoparticles, and
incorporating a variety of imaging and therapeutic functions for
treatment of nervous system injuries. Importantly, the target of
this delivery system is expressed both in mouse and human brain
injuries. Peptides containing the sequence home to, and can
delivery large cargos to, sites of nervous system injury in the
brain without regard to the blood brain barrier. This is likely due
to compromise of the blood brain barrier in nervous system injury.
The targeting and selectively homing are due to the presence of
molecules at the site of nervous system injury to which the peptide
sequence can bind.
[0054] The binding target for the CAQK peptide is a chondroitin
sulfate proteoglycan (CSPG)-rich protein-carbohydrate extracellular
matrix complex that is overexpressed in CNS injuries and is notably
composed of hyaluronic acid, versican, tenascin-R, and hyaluronan
and proteoglycan link protein (Hapln). The targeted CSPG-rich
extracellular matrix complexes generally are extracellular matrix
containing hyaluronic acid, versican, tenascin-R, and Hapln and
exemplified by the matrix of cultured U251 astrocytoma cells. The
overexpression of this complex results in the formation of a
CSPG-rich glial scar, which is a major barrier to regeneration.
This CSPG-rich extracellular matrix complex is also overexpressed
in stroke and other nervous system injuries. Degradation of the
complex is known to improve the outcome of stroke. CAQK-containing
peptides were also shown to the site of injury in an experimental
model of stroke caused by cerebral ischemia. Thus, the discovered
peptides can selectively target sites of nervous system injury,
especially those that overexpress CSPGs.
[0055] Disclosed are peptides, compositions, and methods for
selective targeting nervous system injury, such as brain injury and
stroke injury, sites of glial scar formation, sites where
hyaluronic acid, versican, tenascin-R, and Hapln are being
deposited, and CSPG-rich extracellular matrix complexes. The
disclosed peptides, compositions, and methods are useful for
selectively targeting acute nervous system injury, such as
traumatic brain injury and stroke injury.
[0056] Disclosed are peptides comprising the amino acid sequence
CAQK (SEQ ID NO:4). In some forms, the peptides have any one or
combinations of the following properties: selective homing to a
site of nervous system injury; selective homing to a site of acute
nervous system injury; selective homing to a site of brain injury;
selective homing to a site of acute brain injury; selective homing
to a site of stroke injury; selective homing to a site of acute
stroke injury; specific binding to one or more of versican,
tenascin-R, and Hapln; selective homing to a site of glial scar
formation; selective homing to a site where hyaluronic acid,
versican, tenascin-R, and Hapln are being deposited; and selective
homing to CSPG-rich extracellular matrix complex.
[0057] Disclosed are compositions that include the disclosed
peptide. In some forms, the composition includes the disclosed
peptide and a cargo composition. In some forms of the composition,
the peptide and the cargo composition are covalently coupled or
non-covalently associated with each other. In some forms, the
composition includes the disclosed peptide and a cargo molecule. In
some forms of the composition, the peptide and the cargo molecule
are covalently coupled or non-covalently associated with each
other.
[0058] The disclosed peptides and compositions are useful for
selective targeting nervous system injury, such as brain injury and
stroke injury, sites of glial scar formation, sites where
hyaluronic acid, versican, tenascin-R, and Hapln are being
deposited, and CSPG-rich extracellular matrix complexes. For
example, the disclosed peptides and compositions are useful for
selectively targeting acute nervous system injury, such as
traumatic brain injury and stroke injury. Thus, methods for
selectively targeting a cargo to a site of acute nervous system
injury in a subject are disclosed.
[0059] In some forms, the method involves administering the
disclosed composition to a subject having an acute nervous system
injury. The composition selectively homes to a site of the nervous
system injury in the subject thereby selectively targeting the
cargo composition of the composition to the site of the nervous
system injury.
[0060] It is to be understood that the disclosed method and
compositions are not limited to specific synthetic methods,
specific analytical techniques, or to particular reagents unless
otherwise specified, and, as such, may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to be
limiting.
Materials
[0061] Disclosed are materials, compositions, and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed method and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutation of these compounds may not be explicitly disclosed,
each is specifically contemplated and described herein. For
example, if a cargo molecule is disclosed and discussed and a
number of modifications that can be made to a number of molecules
including the cargo molecule are discussed, each and every
combination and permutation of cargo molecule and the modifications
that are possible are specifically contemplated unless specifically
indicated to the contrary. Thus, if a class of molecules A, B, and
C are disclosed as well as a class of molecules D, E, and F and an
example of a combination molecule, A-D is disclosed, then even if
each is not individually recited, each is individually and
collectively contemplated. Thus, is this example, each of the
combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are
specifically contemplated and should be considered disclosed from
disclosure of A, B, and C; D, E, and F; and the example combination
A-D. Likewise, any subset or combination of these is also
specifically contemplated and disclosed. Thus, for example, the
sub-group of A-E, B-F, and C-E are specifically contemplated and
should be considered disclosed from disclosure of A, B, and C; D,
E, and F; and the example combination A-D. Further, each of the
materials, compositions, components, etc. contemplated and
disclosed as above can also be specifically and independently
included or excluded from any group, subgroup, list, set, etc. of
such materials. These concepts apply to all aspects of this
application including, but not limited to, steps in methods of
making and using the disclosed compositions. Thus, if there are a
variety of additional steps that can be performed it is understood
that each of these additional steps can be performed with any
specific embodiment or combination of embodiments of the disclosed
methods, and that each such combination is specifically
contemplated and should be considered disclosed.
A. CAQK Compositions
[0062] Disclosed are compositions that include the disclosed
peptide. In some forms, the composition includes the disclosed
peptide and a cargo composition. In some forms, the cargo
composition includes one or more cargo molecules. In some forms,
the cargo composition includes a surface molecule. In some forms,
the composition includes the disclosed peptide and a surface
molecule. In some forms, the cargo composition includes a surface
molecule.
[0063] In some forms, the composition can include a plurality of
surface molecules, a plurality of CAQK peptides and a plurality of
cargo molecules. In some forms, the composition can include one or
more surface molecules, a plurality of CAQK peptides and a
plurality of cargo molecules. In some forms, the composition can
include a plurality of surface molecules, one or more CAQK peptides
and a plurality of cargo molecules. In some forms, the composition
can include a plurality of surface molecules, a plurality of CAQK
peptides and one or more cargo molecules. In some forms, the
composition can include one or more surface molecules, one or more
CAQK peptides and a plurality of cargo molecules. In some forms,
the composition can include one or more surface molecules, a
plurality of CAQK peptides and one or more cargo molecules. In some
forms, the composition can include a plurality of surface
molecules, one or more CAQK peptides and one or more cargo
molecules.
[0064] In some forms, the composition can include a plurality of
surface molecules, a plurality of CAQK peptides and a plurality of
cargo compositions. In some forms, the composition can include one
or more surface molecules, a plurality of CAQK peptides and a
plurality of cargo compositions. In some forms, the composition can
include a plurality of surface molecules, one or more CAQK peptides
and a plurality of cargo compositions. In some forms, the
composition can include a plurality of surface molecules, a
plurality of CAQK peptides and one or more cargo compositions. In
some forms, the composition can include one or more surface
molecules, one or more CAQK peptides and a plurality of cargo
compositions. In some forms, the composition can include one or
more surface molecules, a plurality of CAQK peptides and one or
more cargo compositions. In some forms, the composition can include
a plurality of surface molecules, one or more CAQK peptides and one
or more cargo compositions.
[0065] In some forms, the composition can include a surface
molecule, a plurality of CAQK peptides and a plurality of cargo
molecules, where one or more of the CAQK peptides and one or more
of the cargo molecules are associated with the surface molecule. In
some forms, the composition can include a surface molecule, a
plurality of CAQK peptides and a plurality of cargo molecules,
where a plurality of the plurality of CAQK peptides and a plurality
of the plurality of cargo molecules are associated with the surface
molecule. In some forms, the composition can include a surface
molecule, a plurality of CAQK peptides and a plurality of cargo
molecules, where the CAQK peptides and the cargo molecules are
associated with the surface molecule.
[0066] In some forms, the composition can include a surface
molecule, a plurality of CAQK peptides and a plurality of cargo
compositions, where one or more of the CAQK peptides and one or
more of the cargo compositions are associated with the surface
molecule. In some forms, the composition can include a surface
molecule, a plurality of CAQK peptides and a plurality of cargo
compositions, where a plurality of the plurality of CAQK peptides
and a plurality of the plurality of cargo compositions are
associated with the surface molecule. In some forms, the
composition can include a surface molecule, a plurality of CAQK
peptides and a plurality of cargo compositions, where the CAQK
peptides and the cargo compositions are associated with the surface
molecule.
[0067] In some forms, the composition can include a surface
molecule, where the surface molecule is multivalent for CAQK
peptides and cargo molecules. In some forms, the composition can
include a surface molecule, where the surface molecule is
multivalent for CAQK peptides and includes one or more cargo
molecules. In some forms, the composition can include a surface
molecule, where the surface molecule is multivalent for cargo
molecules and includes one or more CAQK peptides.
[0068] In some forms, the composition can include a surface
molecule, where the surface molecule is multivalent for CAQK
peptides and cargo compositions. In some forms, the composition can
include a surface molecule, where the surface molecule is
multivalent for CAQK peptides and includes one or more cargo
compositions. In some forms, the composition can include a surface
molecule, where the surface molecule is multivalent for cargo
compositions and includes one or more CAQK peptides.
[0069] In some forms, the composition can include one or more
copies of the peptide. In some forms, the composition can include
at least 5 copies of the peptide. In some forms, the composition
can include at least 10 copies of the peptide. In some forms, the
composition can include at least 100 copies of the peptide. In some
forms, the composition can include at least 1000 copies of the
peptide. In some forms, the composition can include at least 10,000
copies of the peptide.
[0070] In some forms, the composition can include one or more
copies of the cargo composition. In some forms, the composition can
include at least 5 copies of the cargo composition. In some forms,
the composition can include at least 10 copies of the cargo
composition. In some forms, the composition can include at least
100 copies of the cargo composition. In some forms, the composition
can include at least 1000 copies of the cargo composition. In some
forms, the composition can include at least 10,000 copies of the
cargo composition.
[0071] In some forms, the composition can include one or more
copies of the cargo molecule. In some forms, the composition can
include at least 5 copies of the cargo molecule. In some forms, the
composition can include at least 10 copies of the cargo molecule.
In some forms, the composition can include at least 100 copies of
the cargo molecule. In some forms, the composition can include at
least 1000 copies of the cargo molecule. In some forms, the
composition can include at least 10,000 copies of the cargo
molecule.
[0072] In some forms, the composition can include 1 copy of the
surface molecule. In some forms, the composition can include at
least 1 copy of the surface molecule. In some forms, the
composition can include 2 copies of the surface molecule. In some
forms, the composition can include at least 2 copies of the surface
molecule. In some forms, the composition can include 3 copies of
the surface molecule. In some forms, the composition can include at
least 3 copies of the surface molecule. In some forms, the
composition can include 4 copies of the surface molecule. In some
forms, the composition can include at least 4 copies of the surface
molecule. In some forms, the composition can include 5 copies of
the surface molecule. In some forms, the composition can include at
least 5 copies of the surface molecule. In some forms, the
composition can include 10 copies of the surface molecule. In some
forms, the composition can include at least 10 copies of the
surface molecule.
[0073] In some forms, one or more of the cargo molecules can be
conjugated to one or more of the CAQK peptides. In some forms, the
CAQK peptides can be conjugated with a surface molecule. In some
forms, the cargo molecules can be conjugated with a surface
molecule. In some forms, one or more of the conjugated CAQK
peptides can be indirectly conjugated to a surface molecule via a
linker, one or more of the conjugated cargo molecules can be
indirectly conjugated to the surface molecule via a linker, or
both. In some forms, the composition can further include a
plurality of linkers. In some forms, at least one of the linkers
can include polyethylene glycol. In some forms, one or more of the
conjugated cargo molecules and CAQK peptides can be covalently
coupled. In some forms, one or more of the covalently coupled cargo
molecules and CAQK peptides can include fusion peptides. In some
forms, the CAQK peptides can be conjugated with a surface molecule.
In some forms, one or more of the conjugated CAQK peptides can be
directly conjugated to a surface molecule. In some forms, one or
more of the conjugated CAQK peptides can be indirectly conjugated
to a surface molecule. In some forms, one or more of the CAQK
peptides can be covalently coupled to a surface molecule. In some
forms, one or more of the covalently coupled CAQK peptides can be
directly covalently coupled to a surface molecule. In some forms,
one or more of the covalently coupled CAQK peptides can be
indirectly covalently coupled to a surface molecule. In some forms,
the cargo molecules can be conjugated with a surface molecule. In
some forms, one or more of the conjugated cargo molecules are
directly conjugated to a surface molecule. In some forms, one or
more of the conjugated cargo molecules can be indirectly conjugated
to a surface molecule. In some forms, one or more of the cargo
molecules can be covalently coupled to a surface molecule. In some
forms, one or more of the covalently coupled cargo molecules can be
directly covalently coupled to a surface molecule. In some forms,
one or more of the covalently coupled cargo molecules can be
indirectly covalently coupled to a surface molecule.
[0074] In some forms, one or more of the cargo compositions can be
conjugated to one or more of the CAQK peptides. In some forms, the
CAQK peptides can be conjugated with a surface molecule. In some
forms, the cargo composition includes a surface molecule and one or
more cargo molecules. In some forms, the cargo compositions can be
conjugated with the surface molecule. In some forms, one or more of
the conjugated CAQK peptides can be indirectly conjugated to the
surface molecule via a linker, one or more of the cargo peptides
can be indirectly conjugated to the surface molecule via a linker,
or both. In some forms, the composition can further include a
plurality of linkers. In some forms, at least one of the linkers
can include polyethylene glycol. In some forms, one or more of the
cargo compositions and CAQK peptides can be covalently coupled. In
some forms, one or more of the covalently coupled cargo
compositions and CAQK peptides can include fusion peptides. In some
forms, one or more of the conjugated CAQK peptides can be directly
conjugated to the surface molecule. In some forms, one or more of
the conjugated CAQK peptides can be indirectly conjugated to the
surface molecule. In some forms, one or more of the CAQK peptides
can be covalently coupled to the surface molecule. In some forms,
one or more of the covalently coupled CAQK peptides can be directly
covalently coupled to the surface molecule. In some forms, one or
more of the covalently coupled CAQK peptides can be indirectly
covalently coupled to the surface molecule.
[0075] In some forms, the disclosed compositions can have a
therapeutic effect. In some forms, the therapeutic effect can be
reducing damage of a nervous system injury. In some forms, the
therapeutic effect can be increasing retention of nervous system
function following a nervous system injury. In some forms, the
subject can have one or more sites to be targeted, where the
composition homes to one or more of the sites to be targeted. In
some forms, the subject can have a site of nervous system injury,
where the composition has a therapeutic effect at the site of
nervous system injury.
B. CAQK Peptides
[0076] Disclosed are peptides that specifically home to sites of
nervous system injury. These peptides include the amino acid
sequence CAQK and the CAQK sequence is the only determinant of
homing. These peptides can be referred to as CAQK peptides.
Peptides containing the sequence home to, and can delivery large
cargos to, sites of nervous system injury in the brain without
regard to the blood brain barrier. This is likely due to compromise
of the blood brain barrier in nervous system injury. The targeting
and selectively homing are due to the presence of molecules at the
site of nervous system injury to which the peptide sequence can
bind. Generally, CAQK peptides will have any one or combinations of
the following properties: selective homing to a site of nervous
system injury; selective homing to a site of acute nervous system
injury; selective homing to a site of brain injury; selective
homing to a site of acute brain injury; selective homing to a site
of stroke injury; selective homing to a site of acute stroke
injury; specific binding to one or more of versican, tenascin-R,
and Hapln; selective homing to a site of glial scar formation;
selective homing to a site where hyaluronic acid, versican,
tenascin-R, and Hapln are being deposited; and selective homing to
CSPG-rich extracellular matrix complex.
[0077] In some forms, the peptide is 100 amino acids in length or
less, 50 amino acids in length or less, 30 amino acids in length or
less, 20 amino acids in length or less, 15 amino acids in length or
less, 10 amino acids in length or less, 8 amino acids in length or
less, 6 amino acids in length or less, 5 amino acids in length or
less, or 4 amino acids in length. In some forms, the peptide is
linear. In some forms, the peptide is circular.
[0078] In some forms of the peptide, the amino acid sequence CAQK
(SEQ ID NO:4) is at the C terminal end of the peptide. In some
forms, the peptide consists of the amino acid sequence CAQK (SEQ ID
NO:4). In some forms, the peptide is modified. In some forms, the
peptide is a methylated peptide. In some forms, the peptide
includes a methylated amino acid segment. In some forms, the
peptide is N- or C-methylated in at least one position.
[0079] Disclosed are peptides that include the amino acid sequence
CAQK (SEQ ID NO:4). In some forms, the peptide can be a modified
peptide. In some forms, the peptide can be a methylated peptide. In
some forms, one or more of the methylated peptide can include a
methylated amino acid segment. In some forms, the peptide can be N-
or C-methylated in at least one position.
[0080] CAQK peptides are peptides that include the amino acid
sequence CAQK (SEQ ID NO:4). CAQK peptides can be composed of
standard amino acids with standard peptide linkages or can be
embodied in other than standard amino acids and/or with other than
standard peptide linkages. CAQK peptides can include modifications
to the peptide, amino acids, and/or linkages. Examples of suitable
modifications known to those in the art and are described elsewhere
herein.
[0081] In some forms, the peptide can be comprised in a CAQK
composition. In some forms, the CAQK composition can include one or
more cargo compositions. In some forms, the CAQK composition can
include one or more cargo molecules. In some forms, the peptide can
be comprised in a CAQK conjugate. In some forms, the CAQK conjugate
can include one or more cargo compositions. In some forms, the CAQK
conjugate can include one or more cargo molecules. In some forms,
the composition can include a plurality of cargo compositions. In
some forms, the composition can include a plurality of cargo
molecules. In some forms, the composition can include a plurality
of copies of the peptide.
[0082] The disclosed peptides, including CAQK peptides, can have a
length of up to 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400,
500, 1000 or 2000 residues. In forms, the peptides can have a
length of at least 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,
90, 100 or 200 residues. In some forms, the peptides can have a
length of 7 to 200 residues, 7 to 100 residues, 7 to 90 residues, 7
to 80 residues, 7 to 70 residues, 7 to 60 residues, 7 to 50
residues, 7 to 40 residues, 7 to 30 residues, 7 to 20 residues, 7
to 15 residues, 7 to 10 residues, 8 to 200 residues, 8 to 100
residues, 8 to 90 residues, 8 to 80 residues, 8 to 70 residues, 8
to 60 residues, 8 to 50 residues, 8 to 40 residues, 8 to 30
residues, 8 to 20 residues, 8 to 15 residues, 8 to 10 residues, 9
to 200 residues, 9 to 100 residues, 9 to 90 residues, 9 to 80
residues, 9 to 70 residues, 9 to 60 residues, 9 to 50 residues, 9
to 40 residues, 9 to 30 residues, 9 to 20 residues, 9 to 15
residues, 9 to 10 residues, 10 to 200 residues, 10 to 100 residues,
10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to 60
residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues,
10 to 20 residues, 10 to 15 residues, 15 to 200 residues, 15 to 100
residues, 15 to 90 residues, 15 to 80 residues, 15 to 70 residues,
15 to 60 residues, 15 to 50 residues, 15 to 40 residues, 15 to 30
residues, 15 to 20 residues, 20 to 200 residues, 20 to 100
residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues,
20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30
residues. As used herein, the term "residue" refers to an amino
acid or amino acid analog.
[0083] CAQK peptides can be composed of, for example, amino acids,
amino acid analogs, peptide analogs, amino acid mimetics, peptide
mimetics, etc. Although structures, design, etc. of CAQK peptides
is described herein in terms of amino acids and peptides composed
of amino acids for convenience, it is understood that analogous
analogs, mimetics, modified forms, etc. of amino acids and peptides
can also be used as CAQK peptides and designed using similar
principles.
[0084] Bonds and modifications to amino acids that can reduce or
eliminate protease cleavage at a bond are known and can be used in
the disclosed peptides, including CAQK peptides. For example, the
stability and activity of peptides can be increased by protecting
some of the peptide bonds with N-methylation or C-methylation.
Methylated peptides are peptides containing a non-natural methyl
group. Peptides can be methylated at nitrogens (N-methylated
peptides), carbons (C-methylated peptides), and sulfur
(S-methylated peptides). When a portion of a peptide is methylated
the portion that is methylated can be referred to as a methylated
amino acid segment.
[0085] A variety of chemical modification techniques and moieties
are described in, for example, U.S. Pat. Nos. 5,554,728, 6,869,932,
6,828,401, 6,673,580, 6,552,170, 6,420,339, U.S. Pat. Pub.
2006/0210526 and Intl. Pat. App. WO 2006/136586. Some examples of
such modifications include peptide bond surrogates such as those
described in Cudic and Stawikowski, Peptidomimetics: Fmoc
Solid-Phase Pseudopeptide Synthesis, in Methods in Molecular
Biology, vol. 294, 223-246 (2008), and chemical modifications, such
as maleimide capping, polyethylene glycol (PEG) attachment,
maleidification, acylation, alkylation, esterification, and
amidification, to produce structural analogs of the peptide.
[0086] The disclosed peptides can be made in the form of stabilized
peptides and/or formulated as long-circulating forms. For example,
a polyethylene glycol conjugate can be used. The disclosed peptides
can also be administered over a period of time. For example,
peptides can be delivered with an osmotic pump. This can extend the
permeability of the target cells and tissues. Modified forms of
peptides can be used. For example, peptides can be methylated
(which can stabilize the peptides against proteolysis).
[0087] It is understood that there are numerous amino acid and
peptide analogs which can be incorporated into the disclosed
peptides. For example, there are numerous D amino acids or other
non-natural amino acids which can be used. The opposite
stereoisomers of naturally occurring peptides are disclosed, as
well as the stereo isomers of peptide analogs. These amino acids
can readily be incorporated into polypeptide chains by chemical
synthesis or by charging tRNA molecules with the amino acid of
choice and engineering genetic constructs that utilize, for
example, amber codons, to insert the analog amino acid into a
peptide chain in a site specific way (Albericio, F. (2000).
Solid-Phase Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC
Press; Nilsson B L, Soellner M B, Raines R T (2005). "Chemical
Synthesis of Proteins". Annu. Rev. Biophys. Biomol. Struct. 34:
91-118; Thorson et al., Methods in Molec. Biol. 77:43-73 (1991),
Zoller, Current Opinion in Biotechnology, 3:348-354 (1992); Ibba,
Biotechnology & Genetic Engineering Reviews 13:197-216 (1995),
Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB Tech,
12:158-163 (1994); Ibba and Hennecke, Bio/technology, 12:678-682
(1994) all of which are herein incorporated by reference at least
for material related to amino acid analogs).
[0088] Molecules can be produced that resemble peptides, but which
are not connected via a natural peptide linkage. For example,
linkages for amino acids or amino acid analogs can include
CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2 --, --CH.dbd.CH--
(cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, and
--CHH.sub.2SO. These and others can be found in Spatola, A. F. in
Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins,
B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983);
Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide
Backbone Modifications (general review); Morley, Trends Pharm Sci
(1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res
14:177-185 (1979) (--CH.sub.2NH--, CH.sub.2CH.sub.2--); Spatola et
al. Life Sci 38:1243-1249 (1986) (--CH H.sub.2--S); Hann J. Chem.
Soc Perkin Trans. I 307-314 (1982) (--CH--CH--, cis and trans);
Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (--COCH.sub.2--);
Jennings-White et al. Tetrahedron Lett 23:2533 (1982)
(--COCH.sub.2--); Szelke et al. European Appin, EP 45665 CA (1982):
97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay et al. Tetrahedron.
Lett 24:4401-4404 (1983) (--C(OH)CH.sub.2--); and Hruby Life Sci
31:189-199 (1982) (--CH.sub.2--S--); each of which is incorporated
herein by reference. A particularly useful non-peptide linkage is
--CH.sub.2NH--. It is understood that peptide analogs can have more
than one atom between the bond atoms, such as b-alanine,
g-aminobutyric acid, and the like.
[0089] Amino acid analogs and peptide analogs often have enhanced
or desirable properties, such as, more economical production,
greater chemical stability, enhanced pharmacological properties
(half-life, absorption, potency, efficacy, etc.), altered
specificity (e.g., a broad-spectrum of biological activities),
reduced antigenicity, and others.
[0090] D-amino acids can be used to generate more stable peptides,
because D amino acids are not recognized by peptidases and such.
Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) can be used to generate more stable peptides as
long as activity is preserved. Cysteine residues can be used to
cyclize or attach two or more peptides together. This can be
beneficial to constrain peptides into particular conformations
(Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated
herein by reference).
[0091] The disclosed compositions can include or use the disclosed
CAQK peptides in various forms, including peptides and
peptidomimetics as disclosed. For convenience of expression, in
many places herein the use or inclusion of peptides will be
recited. It is understood that, in such cases, it is considered
that CAQK peptides in various forms can also be used or included in
the same or similar ways as is described in terms of peptides, and
such use and inclusion is specifically contemplated and disclosed
thereby.
[0092] As used herein, the term "peptide" is used broadly to mean
peptides, proteins, fragments of proteins and the like. Peptides
are a class of compounds composed of amino acids chemically bound
together. In general, the amino acids are chemically bound together
via amide linkages (CONH) in peptides. The term "peptidomimetic,"
as used herein, means a peptide-like molecule that has the activity
of the peptide upon which it is structurally based. Such
peptidomimetics include chemically modified peptides, peptide-like
molecules containing non-naturally occurring amino acids, and
peptoids and have an activity such as that from which the
peptidomimetic is derived (see, for example, Goodman and Ro,
Peptidomimetics for Drug Design, in "Burger's Medicinal Chemistry
and Drug Discovery" Vol. 1 (ed. M. E. Wolff; John Wiley & Sons
1995), pages 803-861). In some forms of peptidomimetics, amino
acids can be bound together by chemical bonds other than amide
linkages, as is known in the art. For example, the amino acids may
be bound by amine linkages. The C-terminal end of a peptide refers
to the end of the peptide having the peptide's terminal carboxyl
group. This is the last amino acid of the peptide when considered
from the N to C orientation of the peptide. The CAQK peptide is
further describes and defined elsewhere herein.
[0093] As used herein, the term "non-natural amino acid" refers to
an organic compound that has a structure similar to a natural amino
acid so that it mimics the structure and reactivity of a natural
amino acid. The non-natural amino acid as defined herein generally
increases or enhances the properties of a peptide (e.g.,
selectivity, stability) when the non-natural amino acid is either
substituted for a natural amino acid or incorporated into a
peptide.
[0094] As used herein, the term "homing molecule" means any
molecule that selectively homes in vivo to specific cells or
specific tissue in preference to normal tissue. Similarly, the term
"homing peptide" or "homing peptidomimetic" means a peptide that
selectively homes in vivo to specific cells or specific tissue in
preference to normal tissue. It is understood that a homing
molecule that selectively homes in vivo to specific cells or
specific tissue or can exhibit preferential homing to r specific
cells or specific tissue. The CAQK peptide is an example of a
homing molecule.
[0095] As used herein, "selectively homes" means that, in vivo, the
homing molecule binds preferentially to the target as compared to
non-target. For example, the homing molecule can bind
preferentially to targets at sites of nervous system injury, as
compared to normal or non-injured tissue. Selective homing to, for
example, a site of nervous system injury generally is characterized
by at least a two-fold greater localization at the site of nervous
system injury, as compared to several non-nervous system tissue
types or to non-injured nervous system tissue. A homing molecule
can be characterized by, for example, 5-fold, 10-fold, 20-fold or
more preferential localization to the target as compared to one or
more non-targets. For example, a homing molecule can be
characterized by, for example, 5-fold, 10-fold, 20-fold or more
preferential localization to a site of nervous system injury as
compared to several non-nervous system tissue types or to
non-injured nervous system tissue, or as compared to most or all
non-nervous system tissue. Thus, it is understood that, in some
cases, a homing molecule, such as the CAQK peptide, homes, in part,
to one or more normal organs or one or more normal tissues in
addition to homing to the target tissue. Selective homing can also
be referred to as targeting. The molecules, proteins, cells,
tissues, etc. that are targeted by homing molecules can be referred
to as targets, targeted molecules, proteins, cells, tissues,
etc.
C. Cargo Compositions
[0096] Cargo compositions are molecules or compositions that are
associated with the disclosed peptides in a composition. Doing so
allows the peptide to mediate targeting and homing of the cargo
composition to the target of the peptide (e.g., a site of nervous
system injury). The cargo composition is or contains a cargo that
is desired to be targeted to site of a nervous system injury. The
cargo composition can be a cargo molecule or a plurality of cargo
molecules. Or the cargo composition can be a surface molecule. Or
the cargo composition can include one or more cargo molecules and
one or more other components, such as a surface molecule. In this
way, cargo compositions are generally defined by their components
and their association (and administration) with the disclosed
peptides. Cargo composition can include one or more cargo
molecules. Cargo compositions can include two or more different
cargo molecules.
1. Cargo Molecules
[0097] Cargo molecules are any molecule that is associated with a
disclosed peptide. The association can be direct or indirect and
covalent or non-covalent. Generally, cargo molecules are molecules
that are desired to be targeted to site of a nervous system
injury.
[0098] The disclosed compositions can include one or more cargo
molecules. Generally, the disclosed compositions can include a
plurality of cargo molecules. The disclosed compositions can
include a single type of cargo molecule or a plurality of different
types of cargo molecules. Thus, for example, the disclosed
compositions can include a plurality of different types of cargo
molecules where a plurality of one or more of the different types
of cargo molecules can be present.
[0099] Cargo molecules can be or can include, for example,
therapeutic agents, therapeutic proteins, therapeutic compounds,
therapeutic compositions, polypeptides, nucleic acid molecules,
functional nucleic acids, small molecules, detectable agents,
labels, labeling agents, contrast agents, imaging agents,
fluorophores, fluorescein, rhodamine, radionuclides, Lutetium-177
(.sup.177Lu), Rhenium-188 (.sup.188Re), Gallium-68 (.sup.68Ga),
Yttrium-90 (.sup.90Y), Technetium-99m (.sup.99mTc), Holmium-166
(.sup.166Ho), Iodine-131 (.sup.131I), Indium-111 (.sup.111In),
Flourine-18 (.sup.18F), Carbon-11 (.sup.11C), Carbon-13 (.sup.13C),
Nitrogen-13 (.sup.13N), Oxygen-15 (.sup.15O), Bromine-75
(.sup.75Br), Bromine-76 (.sup.76Br), Iodine-124 (124I), Thalium-201
(.sup.201TI), Technetium-99 (.sup.99Tc), Iodine-123 (.sup.123I).
This is a non-exclusive and a non-inclusive list. Any molecule that
is desired to be targeted to a site of nervous system injury can be
a cargo molecule.
[0100] Cargo molecules can be associated with and arranged in the
compositions in a variety of configurations. In some forms, cargo
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of surface molecules. In some forms, cargo
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of CAQK peptides. In some forms, cargo
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of CAQK peptides, where the CAQK peptides
can be associated with, conjugated to, and/or covalently coupled to
a plurality of surface molecules. Combinations of these
combinations can also be used.
[0101] i. Therapeutic Agents for Nervous System Injuries
[0102] A significant use of the disclosed compositions that target
and home to nervous system injuries is to deliver cargo molecules
to sites of nervous system injury. Therapeutic agents are a
particularly useful cargo molecule to delivery to sites of nervous
system injury. A number of therapeutic agents are known for effects
that would be useful at the sites of nervous system injury, some of
which are described below.
[0103] a. Statins
[0104] Statins form a class of lipid-lowering medications that
inhibit the enzyme HMG-CoA reductase, which plays a central role in
the production of cholesterol. However, statins also exhibit
pleiotropic properties that make them potentially attractive
neuroprotective agents. Preclinical studies demonstrate that
statins target multiple secondary injury pathways and improve
functional outcome after experimental TBI (Wang et al., Exp.
Neurol. 206:59-69 (2007)). Statins decrease apoptosis after trauma
and favorably alter the ratio of anti-apoptotic to apoptotic
factors (Lu et al. J. Neurotrauma 24:1132-1146 (2007)). Studies
show that statins may also promote the growth and differentiation
of new neurons and upregulate neurotrophic factors, including BDNF
and vascular endothelial growth factor (VEGF) (Wu et al., J.
Neurotrauma 25:130-139 (2008); Chen et al., J. Cereb. Blood Flow.
Metab. 25:281-290 (2005)). TBI models have also revealed that
statins limit the production of inflammatory mediators, glial cell
activation and cerebral edema, while increasing the integrity of
the blood brain barrier (Chen et al., J. Cereb. Blood Flow. Metab.
25:281-290 (2005)). However, statins can cause life-threatening
muscle damage known as rhabdomyolysis, which can cause severe
muscle pain, liver damage, kidney failure, and death. Statins are
also known to have effects on the coagulation and fibrinolytic
systems, which may be the basis of their protective effects in
cardiovascular disease (Jansen et al., Crit. Care 17:227
(2013)).
[0105] b. Progesterone
[0106] Progesterone is an endogenous steroid and progestogen sex
hormone, whose receptors are expressed in the CNS of both males and
females. Progesterone has been shown to have neuroprotective
effects in experimental spinal cord injury (SCI), stroke and TBI
(Gonzalez Deniselle et al., J. Steroid Biochem. Mol. Biol.
83:199-209 (2002); Jiang et al., Brain Res. 735:101-107 (1996);
Roof et al., J. Neurotrauma 17:367-388 (2000)). However, recent
reports question its effectiveness in trauma models. A systematic
review of progesterone treatment in CNS injury raised concerns
about the methodological quality of the TBI studies, and
quantitative evaluation revealed possible experimental bias in
these studies (Gibson et al., Brain 131:318-328 (2008)).
Additionally, no protective effects of progesterone were observed
at doses previously reported to be effective by others, in well
characterized models of either TBI or SCI (Fee et al., Brain Res.
1137:146-152 (2007); Gilmer et al., J. Neurotrauma 25:593-602
(2008)). These data suggest that the use of progesterone for
treating TBI is still questionable.
[0107] c. Cyclosporine A
[0108] The early effects of TBI include metabolic crises such as
mitochondrial failure. Mitochondrial failure leads to energy and
ionic imbalances, reduced brain ATP levels, changes in
mitochondrial permeability transition, release of cytochrome c and
induction of pro-apoptotic events (Mazzeo et al., Exp. Neurol.
218:363-370 (2009)). Cyclosporine A (also known as cyclosporin), is
an immunosuppressant drug widely used in organ transplantation to
prevent rejection. Cyclosporine A has been shown to attenuate
mitochondrial failure by binding to cyclophilin D and stabilizing
the mitochondrial permeability transition pore (Szabo et al., J.
Biol. Chem. 266:3376-3379 (1991)). Treatment with Cyclosporine A
reduced axonal damage in diffuse axonal injury models and decreased
lesion size following controlled cortical impact in TBI (Okonkwo et
al., J. Cereb. Blood Flow Metab. 19:443-451 (1999); Sullivan et
al., Neuroscience 101:289-295 (2000)).
[0109] Despite such advantages, Cyclosporine A shows relatively
poor brain penetration, it has a biphasic drug-response curve, and
prolonged use adversely impacts the immune system (Margulies et
al., J. Neurotrauma 26:925-939 (2009)).
[0110] d. Diketopiperazines
[0111] etopiperazines are cyclized dipeptides that were developed
based on the tripeptide thyrotropin-releasing hormone.
Thyrotropin-releasing hormone and its analogs inhibit multiple
secondary injury factors and processes, proving to be highly
neuroprotective in experimental neurotrauma (Pitts et al.,
Neurotrauma 12:235-243 (1995)). Four structurally different
diketopiperazines demonstrated significant neuroprotective
properties both in vitro and in animal TBI studies, one of which
(35b) showed effectiveness across TBI models and species. In
neuronal cell cultures, 35b provided neuroprotection in multiple
models of necrotic and apoptotic cell death, reducing apoptotic
cell death (Faden et al., J. Cereb. Blood Flow Metab. 23:342-354
(2003)). Given their safety profile and their multipotential
neuroprotective effects in experimental TBI models,
diketopiperazines are attractive candidates for TBI therapy.
[0112] e. Substance P Antagonists
[0113] Blood brain barrier disruption is an important contributor
to secondary injury following TBI, and therapies to restore blood
brain barrier functionality are under investigation for
neuroprotection. The localized permeability of the blood brain
barrier and the delayed onset of secondary injury provide a window
of opportunity for therapeutic intervention. The duration of the
blood brain barrier impairment is at least up to five days
(Cunningham et al., J. Neurotrauma 31:505-514 (2014)). Within this
time window, affinity ligand-based (synaphic) targeting can be an
effective drug delivery approach, with results showing as high as
35-fold enhancement in the accumulation of systemically
administered imaging agents and therapeutics at and around the site
of injury.
[0114] Substance P is released early following trauma as part of a
neurogenic inflammatory response. Inhibition of post-traumatic
substance P activity, either by preventing substance P release or
by antagonism of the neurokinin-1 receptor, reduced inflammation
associated with acute TBI and maintained the integrity of the blood
brain barrier (Nimmo et al., Neuropeptides 38:40-47 (2004)).
Furthermore, administration of substance P antagonists decreased
blood brain barrier permeability and edema formation, reduced
axonal injury, enhanced neuronal survival and improved behavioral
outcomes following experimental TBI (Donkin et al., Curr. Opin.
Neurol. 23:293-299 (2010); Donkin et al., J. Cereb. Blood Flow
Metab. 29:1388-1398 (2009)). An evaluation of the therapeutic
window, along with further investigation in additional animal
models and species is needed.
[0115] f. SUR1-regulated NC.sub.ca-ATp Channel Inhibitors
[0116] Edema and progressive secondary hemorrhage are important
secondary injury mechanisms that contribute to neurological
impairments in patients after TBI. Studies suggest that
upregulation of sulfonylurea receptor 1 (SUR1)-regulated
NC.sub.ca-ATp channels in microvascular endothelium plays a key
role in secondary injury pathways (Simard et al., Curr. Opin.
Pharmacol. 8:42-49 (2008)). Glibenclamide, an antidiabetic drug in
the class of medications known as sulfonylureas, also functions as
a SUR1 inhibitor, leading to a reduction in edema, secondary
hemorrhage, inflammation, apoptosis, and lesion size in
experimental TBI and subarachnoid hemorrhage models (Simard et al.,
J. Cereb. Blood Flow Metab. 29:317-330 (2009); Simard et al., J.
Neurotrauma 26:2257-2267 (2009)). Glibenclamide treatment was also
shown to improve functional recovery after TBI (Simard et al., J.
Neurotrauma 26:2257-2267 (2009)).
[0117] g. Cell Cycle Inhibitors
[0118] Upregulation of cell cycle proteins occurs in both mitotic
(astrocytes and microglia) and post-mitotic (neurons,
oligodendroglia) cells after CNS injury, and is associated with
caspase-mediated neuronal apoptosis and glial proliferation after
TBI (Di Giovanni et al., PNAS 102:8333-8338 (2005)). Cell cycle
inhibitors have been extensively evaluated in cancer, but have also
been shown to be strongly neuroprotective in vivo. Inhibitors of
the cell cycle, such as flavopiridol, a semi-synthetic flavonoid,
and the purine analogues roscovitine and olomoucine, exert powerful
neuroprotective effects in various models of neuronal cell death,
as well as inhibitory effects on the proliferation and activation
of astrocytes and microglia (Di Giovanni et al., PNAS 102:8333-8338
(2005); Cernak et al., Cell Cycle 4:1286-1293 (2005); Verdaguer et
al., J. Pharmacol. Exp. Ther. 308:609-616 (2004)).
Intracerebroventricular administration of flavopiridol (an
inhibitor of all major cyclic-dependent kinases) after fluid
percussion injury in rats reduced lesion volume by approximately
70% and improved cognitive and sensorimotor recovery to the level
of uninjured controls. In addition, flavopiridol markedly reduced
glial cell activation, and these changes were associated with
suppression of cell cycle proteins in neurons, astrocytes, and
microglia. Furthermore, delayed administration of flavopiridol had
similar neuroprotective effects; with systemic intraperitoneal
treatment given 24 hours post-trauma causing a significant
reduction in lesion volumes (Cernak et al., Cell Cycle 4:1286-1293
(2005)).
[0119] Roscovitine in particular, is a very selective cell cycle
inhibitor. In addition to improving functional recovery and
mediating a reduction in lesion size, central administration of
roscovitine reduced astrogliosis and produced a marked inhibition
of microglial-mediated neuroinflammation (Hilton et al., J. Cereb.
Blood Flow Metab. 28:1845-1859 (2008)). Protective effects of cell
cycle inhibitors have also been demonstrated after experimental
spinal cord injury and stroke (Byrnes et al., Brain 130:2977-2992
(2007); Osuga et al., PNAS 97:10254-10259 (2000)). However, the
toxicity of these cell cycle inhibitors presents a major
disadvantage.
[0120] h. Cell-Based Therapies
[0121] Endogenous therapeutic stem cell strategies focus on
increasing mobilization, longevity, and production of neural stem
cells in the subventricular zone and dentate gyrus (George et al.,
Neuron 87:297-309 (2015)). While the brain has a limited capacity
for regeneration, endogenous neural stem cells, as well as numerous
pro-regenerative molecules, can participate in replacing and
repairing damaged or diseased neurons and glial cells. One
particular benefit of using endogenous regeneration is the
avoidance of the host immune response.
[0122] Exogenous stem cell treatments refer to transplanted cells
from another source into a patient. Exogenous stem cells have been
delivered to the brain via the blood stream or direct
transplantation and have shown great promise in animal models to
enhance stroke recovery. Exogenous stem cells are typically divided
into three categories: (1) immortalized cell lines, (2) neural
progenitor cells or neural stem cells, and (3) bone marrow-derived
hematopoietic/endothelial progenitors and stromal cells (Bliss et
al., Neurobiol. Dis. 37:275-283 (2010)). Immortalized cell lines
have been developed from tumor cells or from manipulation with
oncogenes. NT2N cells, which are derived from teratocarcinoma,
differentiate into post-mitotic neuron-like cells with the addition
of retinoic acid and mitotic inhibitors, and have been shown to
improve outcome in several ischemic models (Saporta et al.,
Neuroscience 91:519-525 (1999)). ReNeuron's cells, which were
engineered to be immortalized only in the presence of tamoxifen to
reduce the risk of tumor formation, have shown dose-dependent
recovery in stroke rodent models (Stroemer et al., Neurorehabil.
Neural. Repair. 23:895-909 (2009); Stroemer et al., Front Biosci.
13:2290-2292 (2008)).
[0123] Human neural progenitor cells are derived from embryonic and
fetal tissue and have the ability to produce astrocytes, neurons,
and oligodendrocytes (Gage, Science 287:1433-1438 (2000)). In
stroke models, neural progenitor cells are able to migrate to the
injured regions and improve recovery (Zhang et al., Nat.
Biotechnol. 19:1129-1133 (2001)). Progenitor cells derived from
bone marrow, umbilical cord blood and adipose tissue have all been
shown to improve recovery in stroke models (Shen et al., J. Cereb.
Blood Flow Metab. 27:6-13 (2007)).
[0124] The discovery of induced pluripotent stem (iPS) cells
created a paradigm shift in cell therapy, as it allowed researchers
to bypass many of the concerns of traditional stem cell therapy,
such as ethical concerns, supply limitations, and the possible
requirement of immunosuppression (Meissner et al., Nat. Biotechnol.
25:1177-1181 (2007)). Developments in this field have included
vector- and transgene-free techniques to derive iPS cells that
improve functional outcome after brain ischemia (Mohamad et al.,
PLoS One 8:e64160 (2013)).
[0125] However, the precise mechanism of action of stem cell
therapeutics remains elusive, thus limiting the design of trials
and applications.
[0126] i. siRNA Treatment
[0127] Previous studies on siRNA therapy of brain injuries have
either used direct injection into the CNS space or silenced a
target present in the brain endothelial cells (Fukuda et al., Genes
4:435-456 (2013)). A number of targets for gene silencing, such as
Bcl-2 family proteins, caspases, histone deacetylases (HDACs), and
phosphatase and tensin homolog (PTEN), have been suggested for
brain injury treatment.
[0128] The Bcl-2 family of proteins is localized to the outer
membrane of mitochondria, where it can either be pro-apoptotic or
anti-apoptotic, making it an important apoptosis regulator. As
discussed above, secondary injury in TBI can trigger pro-apoptotic
events. Therefore, the use of Bcl-2 inhibitors might be a good
strategy in treating TBI and stroke. Bcl-2 inhibitors include the
antisense oligonucleotide drug Genasense, the small molecule
inhibitor compounds ABT-737 and ABT-263, and the small molecule
oral drug Venetoclax.
[0129] Caspases are a family of protease enzymes that play
essential roles in programmed cell death and inflammation. Other
recently identified roles of caspases include cell proliferation,
tumor suppression, cell differentiation, neural development and
axon guidance and aging (Shalini et al., Cell Death Diff.
22:526-539 (2015)). As excessive programmed cell death is a
prominent feature in neurodegenerative diseases, caspases make a
good target in the treatment of TBI.
[0130] Histone deacetylases (HDAC) are a class of enzymes that
remove acetyl groups from amino acids on histones, allowing the
histones to wrap the DNA more tightly. HDACs are involved in
various gene regulation pathways including signal transduction,
cell cycle, and cancer pathways. Deregulation of histone
modification has been implicated in neurological and psychological
disorders such as Schizophrenia and Huntington's disease (Lee et
al., Neurotherapeutics 10:664-676 (2013)). Accordingly, some
studies suggest that HDAC inhibitors have therapeutic benefits in
various neurological and psychiatric disorders (Grayson et al.,
Mol. Pharm. 77:126-135 (2010)). However, since many neurological
disorders only affect specific brain regions, an understanding of
the specificity of HDACs is imperative before further investigation
for their use in TBI therapy.
[0131] Phosphatase and tensin homolog (PTEN) is a protein that
seems to act primarily as a tumor suppressor. However, its absence
has also been implicated in nerve regeneration in mice, thus making
it an attractive target for inhibition in treating TBI (Liu et al.,
Nat. Neurosci. 13:1075-1081 (2010)).
[0132] ii. Functional Nucleic Acids
[0133] Cargo molecules can be functional nucleic acids. Functional
nucleic acids are nucleic acid molecules that have a specific
function, such as binding a target molecule, serving as an enzyme
substrate or cofactor, or catalyzing a specific reaction. For
example, functional nucleic acids can bind a target nucleic acid
(RNA or DNA) or can serve as enzyme substrate-guiding sequence (or
guide). Functional nucleic acid molecules can be divided into the
following categories, which are not meant to be limiting. For
example, functional nucleic acids include antisense molecules,
aptamers, ribozymes, triplex forming molecules, RNA interference
(RNAi), CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) RNA (crRNA), and external guide sequences. The functional
nucleic acid molecules can act as affectors, inhibitors,
modulators, and stimulators of a specific activity possessed by a
target molecule, or the functional nucleic acid molecules can
possess a de novo activity independent of any other molecules.
[0134] Functional nucleic acid molecules can interact with any
macromolecule, such as DNA, RNA, polypeptides, or carbohydrate
chains. Often functional nucleic acids are designed to interact
with other nucleic acids based on sequence complementarity between
the target molecule and the functional nucleic acid molecule. In
other situations, the specific recognition between the functional
nucleic acid molecule and the target molecule is not based on
sequence complementarity between the functional nucleic acid
molecule and the target molecule, but rather is based on the
formation of tertiary structure that allows specific recognition to
take place.
[0135] Antisense molecules are designed to interact with a target
nucleic acid molecule through either canonical or non-canonical
base pairing. The interaction of the antisense molecule and the
target molecule is designed to promote the destruction of the
target molecule through, for example, RNase H mediated RNA-DNA
hybrid degradation. Alternatively the antisense molecule is
designed to interrupt a processing function that normally would
take place on the target molecule, such as transcription or
replication. Antisense molecules can be designed based on the
sequence of the target molecule. Numerous methods for optimization
of antisense efficiency by finding the most accessible regions of
the target molecule exist. Exemplary methods would be in vitro
selection experiments and DNA modification studies using DMS and
DEPC. It is preferred that antisense molecules bind the target
molecule with a dissociation constant (K.sub.d) less than or equal
to 10.sup.-6, 10.sup.-8, 10.sup.-10, or 10.sup.-12. A
representative sample of methods and techniques which aid in the
design and use of antisense molecules can be found in U.S. Pat.
Nos. 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317,
5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590,
5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522,
6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004,
6,046,319, and 6,057,437.
[0136] Triplex forming functional nucleic acid molecules are
molecules that can interact with either double-stranded or
single-stranded nucleic acid.
[0137] When triplex molecules interact with a target region, a
structure called a triplex is formed, in which there are three
strands of DNA forming a complex dependent on both Watson-Crick and
Hoogsteen base-pairing. Triplex molecules are preferred because
they can bind target regions with high affinity and specificity. It
is preferred that the triplex forming molecules bind the target
molecule with a K.sub.d less than 10-6, 10-8, 10-10, or 10-12.
Representative examples of how to make and use triplex forming
molecules to bind a variety of different target molecules can be
found in U.S. Pat. Nos. 5,176,996, 5,645,985, 5,650,316, 5,683,874,
5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426.
[0138] Gene expression can also be effectively silenced in a highly
specific manner through RNA interference (RNAi). This silencing was
originally observed with the addition of double stranded RNA
(dsRNA) (Fire, A., et al., Nature, 391:806-11 (1998); Napoli, C.,
et al., Plant Cell, 2:279-89 (1990); Hannon, G. J., Nature,
418:244-51 (2002)). Once dsRNA enters a cell, it is cleaved by an
RNase III--like enzyme, Dicer, into double stranded small
interfering RNAs (siRNA) 21-23 nucleotides in length that contain 2
nucleotide overhangs on the 3' ends (Elbashir, S. M., et al., Genes
Dev., 15:188-200 (2001); Bernstein, E., et al., Nature, 409:363-6
(2001); Hammond, S. M., et al., Nature, 404:293-6 (2000)). In an
ATP-dependent step, the siRNAs become integrated into a
multi-subunit protein complex, commonly known as the RNAi induced
silencing complex (RISC), which guides the siRNAs to the target RNA
sequence (Nykanen, A., et al., Cell, 107:309-21 (2001)). At some
point the siRNA duplex unwinds, and it appears that the antisense
strand remains bound to RISC and directs degradation of the
complementary mRNA sequence by a combination of endo and
exonucleases (Martinez, J., et al., Cell, 110:563-74 (2002)).
However, the effect of RNAi or siRNA or their use is not limited to
any type of mechanism.
[0139] Small Interfering RNA (siRNA) is a double-stranded RNA that
can induce sequence-specific post-transcriptional gene silencing,
thereby decreasing or even inhibiting gene expression. In one
example, an siRNA triggers the specific degradation of homologous
RNA molecules, such as mRNAs, within the region of sequence
identity between both the siRNA and the target RNA. For example, WO
02/44321 discloses siRNAs capable of sequence-specific degradation
of target mRNAs when base-paired with 3' overhanging ends, herein
incorporated by reference for the method of making these siRNAs.
Sequence specific gene silencing can be achieved in mammalian cells
using synthetic, short double-stranded RNAs that mimic the siRNAs
produced by the enzyme dicer (Elbashir, S. M., et al., Nature,
411:494 498(2001); Ui-Tei, K., et al., FEBS Lett, 479:79-82
(2000)). siRNA can be chemically or in vitro-synthesized or can be
the result of short double-stranded hairpin-like RNAs (shRNAs) that
are processed into siRNAs inside the cell. Synthetic siRNAs are
generally designed using algorithms and a conventional DNA/RNA
synthesizer. Suppliers include Ambion (Austin, Tex.), ChemGenes
(Ashland, Mass.), Dharmacon (Lafayette, Colo.), Glen Research
(Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo
(Boulder, Colo.), and Qiagen (Vento, The Netherlands). siRNA can
also be synthesized in vitro using kits such as Ambion's
SILENCER.RTM. siRNA Construction Kit.
[0140] Similar to RNAi, CRISPR (Clustered Regularly Interspaced
Short Palindromic Repeats) interference is a powerful approach, via
selective DNA cleavage, for reducing gene expression of
endogenously expressed proteins. CRISPRs are genetic elements
containing direct repeats separated by unique spacers, many of
which are identical to sequences found in phage and other foreign
genetic elements. Recent work has demonstrated the role of CRISPRs
in adaptive immunity and shown that small RNAs derived from CRISPRs
(crRNAs) are implemented as homing oligonucleotides for the
targeted interference of foreign DNA (Jinek et al., Science,
337:816-821 (2012)). crRNAs are used to selectively cleave DNA at
the genetic level.
[0141] Where the functional nucleic acid serves as an enzyme
cofactor, the cofactor can be, for example, a substrate-guiding
sequence (or guide), which directs a nuclease to cleave a substrate
(an RNA or DNA).
[0142] 2. Surface Molecules
[0143] Surface molecules can be used to couple, associate,
encapsulate, hold, etc. components of the disclosed compositions.
Surface molecules can be associated with and arranged in the
compositions in a variety of configurations. In some forms, surface
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of peptides (such as CAQK peptides), a
plurality of cargo molecules, or both. In some forms, surface
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of peptides (such as CAQK peptides), where
the peptides can be associated with, conjugated to, and/or
covalently coupled to a plurality of cargo molecules. In some
forms, surface molecules can be associated with, conjugated to,
and/or covalently coupled to a plurality of cargo molecules, where
the cargo molecules can be associated with, conjugated to, and/or
covalently coupled to a plurality of peptides (such as CAQK
peptides). Combinations of these combinations can also be used.
[0144] The surface molecules, alternatively referred to as a
surface particles, disclosed herein can be associated with CAQK
peptides and cargo molecules in such a way that the composition is
delivered to a target. The surface molecule can be any substance
that can be used with the CAQK peptides and cargo molecules, and is
not restricted by size or substance. The term surface molecule is
used to identify a component of the disclosed composition but is
not intended to be limiting. In particular, the disclosed surface
molecules are not limited to substances, compounds, compositions,
particles or other materials composed of a single molecule. Rather,
the disclosed surface molecules are any substance(s), compound(s),
composition(s), particle(s) and/or other material(s) that can be
associated with one or more CAQK peptides such that at least some
of the CAQK peptides are presented and/or accessible on the surface
of the surface molecule. A variety of examples of suitable surface
molecules are described and disclosed herein.
[0145] In some forms, the surface molecule can include a
nanoparticle, a nanoworm, an iron oxide nanoworm, an iron oxide
nanoparticle, an albumin nanoparticle, a liposome, a micelle, a
phospholipid, a polymer, a microparticle, a bead, a virus, a phage,
a viral particle, a phage particle, a viral capsid, a phage capsid,
a virus-like particle, or a fluorocarbon microbubble.
[0146] The term "nanoparticle" refers to a nanoscale particle with
a size that is measured in nanometers, for example, a nanoscopic
particle that has at least one dimension of less than about 100 nm.
Examples of nanoparticles include paramagnetic nanoparticles,
superparamagnetic nanoparticles, metal nanoparticles, nanoworms,
fullerene-like materials, inorganic nanotubes, dendrimers (such as
with covalently attached metal chelates), nanofibers, nanohoms,
nano-onions, nanorods, nanoropes and quantum dots. A nanoparticle
can produce a detectable signal, for example, through absorption
and/or emission of photons (including radio frequency and visible
photons) and plasmon resonance.
[0147] Microspheres (or microbubbles) can also be used with the
disclosed compositions and methods disclosed. Microspheres
containing chromophores have been utilized in an extensive variety
of applications, including photonic crystals, biological labeling,
and flow visualization in microfluidic channels. See, for example,
Y. Lin, et al., Appl. Phys Lett. 2002, 81, 3134; D. Wang, et al.,
Chem. Mater. 2003, 15, 2724; X. Gao, et al., J. Biomed. Opt. 2002,
7, 532; M. Han, et al., Nature Biotechnology. 2001, 19, 631; V. M.
Pal, et al., Mag. & Magnetic Mater. 1999, 194, 262, each of
which is incorporated by reference in its entirety. Both the
photostability of the chromophores and the monodispersity of the
microspheres can be important.
[0148] Nanoparticles, such as, for example, metal nanoparticles,
metal oxide nanoparticles, or semiconductor nanocrystals can be
incorporated into microspheres. The optical, magnetic, and
electronic properties of the nanoparticles can allow them to be
observed while associated with the microspheres and can allow the
microspheres to be identified and spatially monitored. For example,
the high photostability, good fluorescence efficiency and wide
emission tunability of colloidally synthesized semiconductor
nanocrystals can make them an excellent choice of chromophore.
Unlike organic dyes, nanocrystals that emit different colors (i.e.
different wavelengths) can be excited simultaneously with a single
light source. Colloidally synthesized semiconductor nanocrystals
(such as, for example, core-shell CdSe/ZnS and CdS/ZnS
nanocrystals) can be incorporated into microspheres. The
microspheres can be monodisperse silica microspheres.
[0149] The nanoparticle can be a metal nanoparticle, a metal oxide
nanoparticle, or a semiconductor nanocrystal. The metal of the
metal nanoparticle or the metal oxide nanoparticle can include
titanium, zirconium, hafnium, vanadium, niobium, tantalum,
chromium, molybdenum, tungsten, manganese, technetium, rhenium,
iron, ruthenium, osmium, cobalt, rhodium, iridium, nickel,
palladium, platinum, copper, silver, gold, zinc, cadmium, scandium,
yttrium, lanthanum, a lanthanide series or actinide series element
(e.g., cerium, praseodymium, neodymium, promethium, samarium,
europium, gadolinium, terbium, dysprosium, holmium, erbium,
thulium, ytterbium, lutetium, thorium, protactinium, and uranium),
boron, aluminum, gallium, indium, thallium, silicon, germanium,
tin, lead, antimony, bismuth, polonium, magnesium, calcium,
strontium, and barium. In some forms, the metal can be iron,
ruthenium, cobalt, rhodium, nickel, palladium, platinum, silver,
gold, cerium or samarium. The metal oxide can be an oxide of any of
these materials or combination of materials. For example, the metal
can be gold, or the metal oxide can be an iron oxide, a cobalt
oxide, a zinc oxide, a cerium oxide, or a titanium oxide.
Preparation of metal and metal oxide nanoparticles is described,
for example, in U.S. Pat. Nos. 5,897,945 and 6,759,199, each of
which is incorporated by reference in its entirety.
[0150] The nanoparticles can be comprised of cargo molecules and a
carrier protein (such as albumin). Such nanoparticles are useful,
for example, to deliver hydrophobic or poorly soluble compounds.
Nanoparticles of poorly water soluble drugs (such as taxane) have
been disclosed in, for example, U.S. Pat. Nos. 5,916,596;
6,506,405; and 6,537,579 and also in U.S. Pat. Pub. No.
2005/0004002A1.
[0151] In some forms, the nanoparticles can have an average or mean
diameter of no greater than about 1000 nanometers (nm), such as no
greater than about any of 900, 800, 700, 600, 500, 400, 300, 200,
and 100 nm. In some forms, the average or mean diameters of the
nanoparticles can be no greater than about 200 nm. In some forms,
the average or mean diameters of the nanoparticles can be no
greater than about 150 nm. In some forms, the average or mean
diameters of the nanoparticles can be no greater than about 100 nm.
In some forms, the average or mean diameter of the nanoparticles
can be about 20 to about 400 nm. In some forms, the average or mean
diameter of the nanoparticles can be about 40 to about 200 nm. In
some forms, the nanoparticles are sterile-filterable.
[0152] The nanoparticles can be present in a dry formulation (such
as lyophilized composition) or suspended in a biocompatible medium.
Suitable biocompatible media include, but are not limited to,
water, buffered aqueous media, saline, buffered saline, optionally
buffered solutions of amino acids, optionally buffered solutions of
proteins, optionally buffered solutions of sugars, optionally
buffered solutions of vitamins, optionally buffered solutions of
synthetic polymers, lipid-containing emulsions, and the like.
[0153] Examples of suitable carrier proteins include proteins
normally found in blood or plasma, which include, but are not
limited to, albumin, immunoglobulin including IgA, lipoproteins,
apolipoprotein B, alpha-acid glycoprotein, beta-2-macroglobulin,
thyroglobulin, transferin, fibronectin, factor VII, factor VIII,
factor IX, factor X, and the like. In some forms, the carrier
protein is non-blood protein, such as casein, .alpha.-lactalbumin,
and beta-lactoglobulin. The carrier proteins may either be natural
in origin or synthetically prepared. In some forms, the
pharmaceutically acceptable carrier includes albumin, such as human
serum albumin. Human serum albumin (HSA) is a highly soluble
globular protein of M.sub.r 65K and consists of 585 amino acids.
HSA is the most abundant protein in the plasma and accounts for
70-80% of the colloid osmotic pressure of human plasma. The amino
acid sequence of HSA contains a total of 17 disulphide bridges, one
free thiol (Cys 34), and a single tryptophan (Trp 214). Intravenous
use of HSA solution has been indicated for the prevention and
treatment of hypovolumic shock (see, e.g., Tullis, JAMA
237:355-360, 460-463 (1977)) and Houser et al., Surgery, Gynecology
and Obstetrics, 150:811-816 (1980)) and in conjunction with
exchange transfusion in the treatment of neonatal
hyperbilirubinemia (see, e.g., Finlayson, Seminars in Thrombosis
and Hemostasis, 6:85-120 (1980)). Other albumins are contemplated,
such as bovine serum albumin. Use of such non-human albumins could
be appropriate, for example, in the context of use of these
compositions in non-human mammals, such as the veterinary
(including domestic pets and agricultural context).
[0154] Carrier proteins (such as albumin) in the composition
generally serve as a carrier for the hydrophobic cargo molecules,
i.e., the carrier protein in the composition makes the cargo
molecules more readily suspendable in an aqueous medium or helps
maintain the suspension as compared to compositions not comprising
a carrier protein. This can avoid the use of toxic solvents (or
surfactants) for solubilizing the cargo molecules, and thereby can
reduce one or more side effects of administration of the cargo
molecules into an individual (such as a human). Thus, in some
forms, the composition described herein can be substantially free
(such as free) of surfactants, such as Cremophor (including
Cremophor EL.RTM. (BASF)). In some forms, the composition can be
substantially free (such as free) of surfactants. A composition is
"substantially free of Cremophor" or "substantially free of
surfactant" if the amount of Cremophor or surfactant in the
composition is not sufficient to cause one or more side effect(s)
in an individual when the composition is administered to the
individual.
[0155] The amount of carrier protein in the composition described
herein will vary depending on other components in the composition.
In some forms, the composition can include a carrier protein in an
amount that is sufficient to stabilize the cargo molecules in an
aqueous suspension, for example, in the form of a stable colloidal
suspension (such as a stable suspension of nanoparticles). In some
forms, the carrier protein is in an amount that reduces the
sedimentation rate of the cargo molecules in an aqueous medium. For
particle-containing compositions, the amount of the carrier protein
also depends on the size and density of nanoparticles of the cargo
molecules.
[0156] Methods of making nanoparticle compositions are known in the
art. For example, nanoparticles containing cargo molecules and
carrier protein (such as albumin) can be prepared under conditions
of high shear forces (e.g., sonication, high pressure
homogenization, or the like). These methods are disclosed in, for
example, U.S. Pat. Nos. 5,916,596; 6,506,405; and 6,537,579 and
also in U.S. Pat. Pub. No. 2005/0004002A1.
[0157] Briefly, the hydrophobic carrier molecules can be dissolved
in an organic solvent, and the solution can be added to a human
serum albumin solution. The mixture is subjected to high pressure
homogenization. The organic solvent can then be removed by
evaporation. The dispersion obtained can be further lyophilized.
Suitable organic solvent include, for example, ketones, esters,
ethers, chlorinated solvents, and other solvents known in the art.
For example, the organic solvent can be methylene chloride and
chloroform/ethanol (for example with a ratio of 1:9, 1:8, 1:7, 1:6,
1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or
9:1).
[0158] The nanoparticle can also be, for example, a heat generating
nanoshell. As used herein, "nanoshell" is a nanoparticle having a
discrete dielectric or semi-conducting core section surrounded by
one or more conducting shell layers. U.S. Pat. No. 6,530,944 is
hereby incorporated by reference herein in its entirety for its
teaching of the methods of making and using metal nanoshells. CAQK
peptides can be attached to the disclosed compositions and/or
carriers.
[0159] "Liposome" as the term is used herein refers to a structure
comprising an outer lipid bi- or multi-layer membrane surrounding
an internal aqueous space. Liposomes can be used to package any
cargo molecule for targeting to nervous system injury. Materials
and procedures for forming liposomes are well-known to those
skilled in the art. Upon dispersion in an appropriate medium, a
wide variety of phospholipids swell, hydrate and form multilamellar
concentric bilayer vesicles with layers of aqueous media separating
the lipid bilayers. These systems are referred to as multilamellar
liposomes or multilamellar lipid vesicles ("MLVs") and have
diameters within the range of 10 nm to 100 .mu.m. These MLVs were
first described by Bangham, et al., J Mol. Biol. 13:238-252 (1965).
In general, lipids or lipophilic substances are dissolved in an
organic solvent. When the solvent is removed, such as under vacuum
by rotary evaporation, the lipid residue forms a film on the wall
of the container. An aqueous solution that typically contains
electrolytes or hydrophilic biologically active materials is then
added to the film. Large MLVs are produced upon agitation. When
smaller MLVs are desired, the larger vesicles are subjected to
sonication, sequential filtration through filters with decreasing
pore size or reduced by other forms of mechanical shearing. There
are also techniques by which MLVs can be reduced both in size and
in number of lamellae, for example, by pressurized extrusion
(Barenholz, et al., FEBS Lett. 99:210-214 (1979)).
[0160] Liposomes can also take the form of unilamnellar vesicles,
which are prepared by more extensive sonication of MLVs, and
consist of a single spherical lipid bilayer surrounding an aqueous
solution. Unilamellar vesicles ("ULVs") can be small, having
diameters within the range of 20 to 200 nm, while larger ULVs can
have diameters within the range of 200 nm to 2.mu.m. There are
several well-known techniques for making unilamellar vesicles. In
Papahadjopoulos, et al., Biochim et Biophys Acta 135:624-238
(1968), sonication of an aqueous dispersion of phospholipids
produces small ULVs having a lipid bilayer surrounding an aqueous
solution. Schneider, U.S. Pat. No. 4,089,801 describes the
formation of liposome precursors by ultrasonication, followed by
the addition of an aqueous medium containing amphiphilic compounds
and centrifugation to form a biomolecular lipid layer system.
[0161] Small ULVs can also be prepared by the ethanol injection
technique described by Batzri et al., Biochim et Biophys Acta
298:1015-1019 (1973) and the ether injection technique of Deamer et
al., Biochim et Biophys Acta 443:629-634 (1976). These methods
involve the rapid injection of an organic solution of lipids into a
buffer solution, which results in the rapid formation of
unilamellar liposomes. Another technique for making ULVs is taught
by Weder et al. in "Liposome Technology", ed. G. Gregoriadis, CRC
Press Inc., Boca Raton, Fla., Vol. I, Chapter 7, pg. 79-107 (1984).
This detergent removal method involves solubilizing the lipids and
additives with detergents by agitation or sonication to produce the
desired vesicles.
[0162] Papahadjopoulos et al., U.S. Pat. No. 4,235,871, describes
the preparation of large ULVs by a reverse phase evaporation
technique that involves the formation of a water-in-oil emulsion of
lipids in an organic solvent and the drug to be encapsulated in an
aqueous buffer solution. The organic solvent is removed under
pressure to yield a mixture which, upon agitation or dispersion in
an aqueous media, is converted to large ULVs. Suzuki et al., U.S.
Pat. No. 4,016,100, describes another method of encapsulating
agents in unilamellar vesicles by freezing/thawing an aqueous
phospholipid dispersion of the agent and lipids.
[0163] In addition to the MLVs and ULVs, liposomes can also be
multivesicular. Described in Kim et al., Biochim et Biophys Acta
728:339-348 (1983), these multivesicular liposomes are spherical
and contain internal granular structures. The outer membrane is a
lipid bilayer and the internal region contains small compartments
separated by bilayer septum. Still yet another type of liposomes
are oligolamellar vesicles ("OLVs"), which have a large center
compartment surrounded by several peripheral lipid layers. These
vesicles, having a diameter of 2-15 .mu.m, are described in Callo,
et al., Cryobiology 22(3):251-267 (1985).
[0164] Mezei et al., U.S. Pat. Nos. 4,485,054 and 4,761,288 also
describe methods of preparing lipid vesicles. More recently, Hsu,
U.S. Pat. No. 5,653,996 describes a method of preparing liposomes
utilizing aerosolization and Yiournas, et al., U.S. Pat. No.
5,013,497 describes a method for preparing liposomes utilizing a
high velocity-shear mixing chamber. Methods are also described that
use specific starting materials to produce ULVs (Wallach, et al.,
U.S. Pat. No. 4,853,228) or OLVs (Wallach, U.S. Pat. Nos. 5,474,848
and 5,628,936).
[0165] A comprehensive review of all the aforementioned lipid
vesicles and methods for their preparation are described in
"Liposome Technology", ed. G. Gregoriadis, CRC Press Inc., Boca
Raton, Fla., Vol. I, II & III, 3.sup.rd Ed. (2006). This and
the aforementioned references describing various lipid vesicles
suitable for use in the disclosed compositions are incorporated
herein by reference.
[0166] "Micelle" as used herein refers to a structure comprising an
outer lipid monolayer. Micelles can be formed in an aqueous medium
when the Critical Micelle Concentration (CMC) is exceeded. Small
micelles in dilute solution at approximately the critical micelle
concentration (CMC) are generally believed to be spherical.
However, under other conditions, they may be in the shape of
distorted spheres, disks, rods, lamellae, and the like. Micelles
formed from relatively low molecular weight amphiphile molecules
can have a high CMC so that the formed micelles dissociate rather
rapidly upon dilution. If this is undesired, amphiphile molecules
with large hydrophobic regions can be used. For example, lipids
with a long fatty acid chain or two fatty acid chains, such as
phospholipids and sphingolipids, or polymers, specifically block
copolymers, can be used.
[0167] Polymeric micelles have been prepared that exhibit CMCs as
low as 10.sup.-6 M (molar). Thus, they tend to be very stable while
at the same time showing the same beneficial characteristics as
amphiphile micelles. Any micelle-forming polymer can be used in the
disclosed compositions and methods. Examples of micelle-forming
polymers include, without limitation, methoxy poly(ethylene
glycol)-b-poly(.epsilon.-caprolactone), conjugates of poly(ethylene
glycol) with phosphatidyl-ethanolamine, poly(ethylene
glycol)-b-polyesters, poly(ethylene glycol)-b-poly(L-aminoacids),
poly(N-vinylpyrrolidone)-b1-poly(orthoesters),
poly(N-vinylpyrrolidone)-b-polyanhydrides and
poly(N-vinylpyrrolidone)-b-poly(alkyl acrylates).
[0168] Micelles can be produced by processes conventional in the
art. Examples of such are described in, for example, Liggins
(Liggins and Burt, Adv. Drug Del. Rev. 54: 191-202, (2002)); Zhang
et al. (Zhang, X. et al., Int. J. Pharm. 132: 195-206, (1996)); and
Churchill (Churchill and Hutchinson, U.S. Pat. No. 4,745,160
(1988)). In one such method, polyether-polyester block copolymers,
which are amphipathic polymers having hydrophilic (polyether) and
hydrophobic (polyester) segments, are used as micelle forming
carriers.
[0169] Another type of micelle can be formed using, for example,
AB-type block copolymers having both hydrophilic and hydrophobic
segments, as described in, for example, Tuzar (Tuzar and
Kratochvil, Adv. Colloid Interface Sci. 6:201-232, (1976)); and
Wilhelm et al. (Wilhelm et al., Macromolecules 24: 1033-1040
(1991)). These polymeric micelles are able to maintain satisfactory
aqueous stability. These micelles, in the range of
approximately<200 nm in size, are effective in reducing
non-selective RES scavenging and show enhanced permeability and
retention.
[0170] U.S. Pat. No. 5,929,177 to Kataoka et al. describes a
polymeric molecule usable as, for example, a drug delivery carrier.
The micelle is formed from a block copolymer having functional
groups on both of its ends and which includes
hydrophilic/hydrophobic segments. The polymer functional groups on
the ends of the block copolymer include amino, carboxyl and
mercapto groups on the .alpha.-terminal and hydroxyl, carboxyl
group, aldehyde group and vinyl group on the .omega.-terminal. The
hydrophilic segment includes polyethylene oxide, while the
hydrophobic segment is derived from lactide, lactone or
(meth)acrylic acid ester.
[0171] Poly(D,L-lactide)-b-methoxypolyethylene glycol (MePEG:PDLLA)
diblock copolymers can be made using MePEG 1900 and 5000. The
reaction can be allowed to proceed for 3 hr at 160.degree. C.,
using stannous octoate (0.25%) as a catalyst. However, a
temperature as low as 130.degree. C. can be used if the reaction is
allowed to proceed for about 6 hours, or a temperature as high as
190.degree. C. can be used if the reaction is carried out for only
about 2 hours.
[0172] N-isopropylacrylamide ("IPAAm") (Kohjin, Tokyo, Japan) and
dimethylacrylamide ("DMAAm") (Wako Pure Chemicals, Tokyo, Japan)
can be used to make hydroxyl-terminated poly(IPAAm-co-DMAAm) in a
radical polymerization process, using the method of Kohori et al.
(1998). (Kohori et al., J. Control. Rel. 55: 87-98, (1998)). The
obtained copolymer can be dissolved in cold water and filtered
through two ultrafiltration membranes with a 10,000 and 20,000
molecular weight cut-off. The polymer solution is first filtered
through a 20,000 molecular weight cut-off membrane. Then the
filtrate was filtered again through a 10,000 molecular weight
cut-off membrane. Three molecular weight fractions can be obtained
as a result, a low molecular weight, a middle molecular weight, and
a high molecular weight fraction. A block copolymer can then be
synthesized by a ring opening polymerization of D,L-lactide from
the terminal hydroxyl group of the poly(IPAAm-co-DMAAm) of the
middle molecular weight fraction. The resulting
poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) copolymer can be purified
as described in Kohori et al. (1999). (Kohori et al., Colloids
Surfaces B: Biointerfaces 16: 195-205, (1999)).
[0173] Examples of block copolymers from which micelles can be
prepared are found in U.S. Pat. No. 5,925,720, to Kataoka et al.,
U.S. Pat. No. 5,412,072 to Sakurai et al., U.S. Pat. No. 5,410,016
to Kataoka et al., U.S. Pat. No. 5,929,177 to Kataoka et al., U.S.
Pat. No. 5,693,751 to Sakurai et al., U.S. Pat. No. 5,449,513 to
Yokoyama et al., WO 96/32434, WO 96/33233 and WO 97/0623, the
contents of all of which are incorporated by reference.
Modifications thereof, which are prepared by introducing thereon a
suitable functional group (including an ethyleneically unsaturated
polymerizable group), are also examples of block copolymers from
which micelles can be prepared. If the block copolymer has a sugar
residue on one end of the hydrophilic polymer segment, as in the
block copolymer of WO 96/32434, the sugar residue should preferably
be subjected to Malaprade oxidation so that a corresponding
aldehyde group may be formed.
[0174] Lipids are synthetically or naturally-occurring molecules,
which includes fats, waxes, sterols, prenol lipids, fat-soluble
vitamins (such as vitamins A, D, E and K), glycerolipids,
monoglycerides, diglycerides, triglycerides, glycerophospholipids,
sphingolipids, phospholipids, fatty acids monoglycerides,
saccharolipids and others. Lipids can be hydrophobic or amphiphilic
small molecules; the amphiphilic nature of some lipids allows them
to form structures such as monolayers, vesicles, micelles,
liposomes, bi-layers or membranes in an appropriate environment
i.e. aqueous environment. Any of a number of lipids can be used as
amphiphile molecules, including amphipathic, neutral, cationic, and
anionic lipids. Such lipids can be used alone or in combination,
and can also include bilayer stabilizing components such as
polyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017, "Polyimide
Oligomers", by Ansell), peptides, proteins, detergents,
lipid-derivatives, such as PEG coupled to phosphatidylethanolamine
and PEG conjugated to ceramides (see U.S. Pat. No. 5,885,613). In
some forms, cloaking agents, which reduce elimination of liposomes
by the host immune system, can also be included, such as
polyamide-oligomer conjugates, e.g., ATTA-lipids, (see U.S. Pat.
No. 5,693,463), and PEG-lipid conjugates (see U.S. Pat. Nos.
5,820,873, 5,534,499 and 5,885,613).
[0175] Any of a number of neutral lipids can be included, referring
to any of a number of lipid species which exist either in an
uncharged or neutral zwitterionic form at physiological pH,
including diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin,
cholesterol, cerebrosides, and diacylglycerols.
[0176] Cationic lipids, which carry a net positive charge at
physiological pH, can readily be used as amphiphile molecules. Such
lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy) propyl-N,N-N-triethylammonium chloride
("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP");
3.beta.-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
("DC-Chol"),
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
-ammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl
carboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine
("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"), and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"). Additionally, a number of commercial
preparations of cationic lipids can be used, such as LIPOFECTIN
(including DOTMA and DOPE, available from GIBCO/BRL), LIPOFECTAMINE
(comprising DOSPA and DOPE, available from GIBCO/BRL), and
TRANSFECTAM (comprising DOGS, in ethanol, from Promega Corp.).
[0177] Anionic lipids can be used as amphiphile molecules and
include, but are not limited to, phosphatidylglycerol, cardiolipin,
diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl
phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,
N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and
other anionic modifying groups joined to neutral lipids.
[0178] Amphiphatic lipids can also be suitable amphiphile
molecules. "Amphipathic lipids" refer to any suitable material,
where the hydrophobic portion of the lipid material orients into a
hydrophobic phase, while the hydrophilic portion orients toward the
aqueous phase. Such compounds include, but are not limited to,
fatty acids, phospholipids, aminolipids, and sphingolipids.
Representative phospholipids include sphingomyelin,
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidic acid, palmitoyloleoyl
phosphatdylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or
dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds,
such as sphingolipids, glycosphingolipid families, diacylglycerols,
and .beta.-acyloxyacids, can also be used. Additionally, such
amphipathic lipids can be readily mixed with other lipids, such as
triglycerides and sterols. Zwitterionic lipids are a form of
amphiphatic lipid.
[0179] Sphingolipids are fatty acids conjugated to the aliphatic
amino alcohol sphingosine. The fatty acid can be covalently bond to
sphingosine via an amide bond. Any amino acid as described above
can be covalently bond to sphingosine to form a sphingolipid. A
sphingolipid can be further modified by covalent bonding through
the a-hydroxyl group. The modification can include alkyl groups,
alkenyl groups, alkynyl groups, aromatic groups, heteroaromatic
groups, cyclyl groups, heterocyclyl groups, phosphonic acid groups.
Non-limiting examples of shingolipids are N-acylsphingosine,
N-Acylsphingomyelin, Forssman antigen.
[0180] Saccharolipids are compounds that contain both fatty acids
and sugars. The fatty acids are covalently bonded to a sugar
backbone. The sugar backbone can contain one or more sugars. The
fatty acids can bond to the sugars via either amide or ester bonds.
The sugar can be any sugar base. The fatty acid can be any fatty
acid as described elsewhere herein. The provided compositions can
include either natural or synthetic saccharolipids. Non-limiting
saccharolipids are UDP-3-O-((.beta.-hydroxymyristoyl)-GlcNAc, lipid
IV A, Kdo2-lipid A.
D. Homing Molecules
[0181] Disclosed are homing molecules that selectively home to
CSPG-rich extracellular matrix complexes (extracellular matrix
containing hyaluronic acid, versican, tenascin-R, and Hapln and
exemplified by the matrix of cultured U251 astrocytoma cells). CAQK
peptides are a type of such homing molecules, but other peptides,
molecules, compounds can also home to CSPG-rich extracellular
matrix complexes at sites of nervous system injury. Such homing
molecules can be identified by any suitable method. For example, in
some forms, the homing molecules can be identified by bringing into
contact the homing molecule and versican, tenascin-R, Hapln, or
combinations thereof, and assessing whether the homing molecule
specifically binds to the versican, tenascin-R, Hapln, or
combination thereof. The homing molecule is identified if the
homing molecule specifically binds to the versican, tenascin-R,
Hapln, or combination thereof. In some forms, the homing molecules
can be identified by bringing into contact the homing molecule and
CSPG-rich extracellular matrix complexes, and assessing whether the
homing molecule specifically binds to the CSPG-rich extracellular
matrix complexes. The homing molecule is identified if the homing
molecule specifically binds to the CSPG-rich extracellular matrix
complexes. In some forms the CSPG-rich extracellular matrix
complexes can be matrix produced by U251 astrocytoma cells.
Methods
A. Methods of Making
[0182] The disclosed peptides, cargo molecules, surface molecules,
cargo compositions, and other components of the disclosed
compositions can be synthesized and produced generally using known
methods, some of which are described elsewhere herein. For example,
methods for peptide synthesis are well known and can be used to
make the disclosed peptides. Cargo molecules, which are often
therapeutic or detectable agents, can be made as is known and/or as
is appropriate for the molecule involved.
[0183] Association of the components of the disclosed compositions
can be aided or accomplished via molecules, conjugates and/or
compositions. Where such molecules, conjugates and/or compositions
are other than CAQK peptides, cargo compositions, cargo molecules,
or surface molecules, they can be referred to herein as linkers.
Such linkers can be any molecule, conjugate, composition, etc. that
can be used to associate components of the disclosed compositions.
Generally, linkers can be used to associate components other than
surface molecules to surface molecules and to associate different
components with each other. Useful linkers include materials that
are biocompatible, have low bioactivity, have low antigenicity,
etc. That is, such useful linker materials can serve the
linking/association function without adding unwanted bioreactivity
to the disclosed compositions. Many such materials are known and
used for similar linking and association functions. Polymer
materials are a particularly useful form of linker material. For
example, polyethylene glycols can be used.
[0184] Disclosed are linkers for associating components of the
disclosed compositions. Such linkers can be any molecule,
conjugate, composition, etc. that can be used to associate
components of the disclosed compositions. Generally, linkers can be
used to associate components other than surface molecules to
surface molecules and to associate different components with each
other. Useful linkers include materials that are biocompatible,
have low bioactivity, have low antigenicity, etc. That is, such
useful linker materials can serve the linking/association function
without adding unwanted bioreactivity to the disclosed
compositions. Many such materials are known and used for similar
linking and association functions. Polymer materials are a
particularly useful form of linker material. For example,
polyethylene glycols can be used.
[0185] Linkers of different lengths can be used to couple the
disclosed components to surface molecules and to each other. A
flexible linker can function well even if relatively short, while a
stiffer linker can be longer to allow effective exposure and
density. The length of a linker can refer to the number of atoms in
a continuous covalent chain between the attachment points on the
components being linked or to the length (in nanometers, for
example) of a continuous covalent chain between the attachment
points on the components being linked. Unless the context clearly
indicates otherwise, the length refers to the shortest continuous
covalent chain between the attachment points on the components
being linked not accounting for side chains, branches, or loops.
Due to flexibility of the linker, all of the linkers may not have
same distance from the surface molecule or between components. Thus
linkers with different chain lengths can make the resulting
composition more effective (by increasing density, for example).
Branched linkers bearing multiple components also allow attachment
of more than one component at a given site of the surface molecule
or on another component. Useful lengths for linkers include at
least, up to, about, exactly, or between 10, 15, 20, 25, 30, 35,
40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000,
and 10,000 atoms. Useful lengths for linkers include at least, up
to, about, exactly, or between 10, 15, 20, 25, 30, 35, 40, 45, 50,
60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,
2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000
nanometers. Any range of these lengths and all lengths between the
listed lengths are specifically contemplated.
[0186] Hydrophilic or water-solubility linkers can increase the
mobility of the attached components. Examples of water-soluble,
biocompatible polymers which can serve as linkers include, but are
not limited to polymers such polyethylene glycol (PEG),
polyethylene oxide (PEO), polyvinyl alcohol, polyhydroxyethyl
methacrylate, polyacrylamide, and natural polymers such as
hyaluronic acid, chondroitin sulfate, carboxymethylcellulose, and
starch. Useful forms of branched tethers include star PEO and comb
PEO. Star PEO can be formed of many PEO "arms" emanating from a
common core.
[0187] Polyethylene glycols (PEGs) are simple, neutral polyethers
which have been given much attention in biotechnical and biomedical
applications (Milton Harris, J. (ed) "Poly(ethylene glycol)
chemistry, biotechnical and biomedical applications" Plenum Press,
New York, 1992). PEGs are soluble in most solvents, including
water, and are highly hydrated in aqueous environments, with two or
three water molecules bound to each ethylene glycol segment; this
hydration phenomenon has the effect of preventing adsorption either
of other polymers or of proteins onto PEG-modified surfaces.
Furthermore, PEGs may readily be modified and bound to other
molecules with only little effect on their chemistry. Their
advantageous solubility and biological properties are apparent from
the many possible uses of PEGs and copolymers thereof, including
block copolymers such as PEG-polyurethanes and PEG-polypropylenes.
Appropriate molecular weights for PEG linkers used in the disclosed
compositions can be from about 120 daltons to about 20 kilodaltons.
For example, PEGs can be at least, up to, about, exactly, or
between 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,
900, 1000, 1200, 1400, 1500, 1600, 1800, 2000, 2500, 3000, 3500,
4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000,
9500, 10,000, 20,000, 30,000, 40,000, and 50,000 daltons. Any range
of these masses and all masses between the listed masses are
specifically contemplated. PEGs are usually available as mixtures
of somewhat heterogeneous masses with a stated average mass
(PEG-5000, for example).
[0188] The disclosed compositions can be produced using any
suitable techniques. Many techniques, reactive groups, chemistries,
etc. for linking components of the types disclosed herein are known
and can be used with the disclosed components and compositions.
[0189] Protein crosslinkers that can be used to crosslink the
disclosed compositions, surface molecules, CAQK peptides, cargo
molecules, cargo compositions, and other molecules are known in the
art and are defined based on utility and structure. Examples
include DSS (Disuccinimidylsuberate), DSP
(Dithiobis(succinimidylpropionate)), DTSSP (3,3'-Dithiobis
(sulfosuccinimidylpropionate)), SULFO BS OCOES
(Bis[2-(sulfosuccinimdooxycarbonyloxy) ethyl]sulfone), BS OCOES
(Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST
(Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO
EGS (Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethylene
glycolbis(sulfosuccinimidylsuccinate)), DPDPB
(1,2-Di[3'-(2'-pyridyldithio) propionamido]butane), BSSS
(Bis(sulfosuccinimdyl) suberate), SMPB
(Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB
(Sulfosuccinimdyl-4-(p-maleimidophenyl) butyrate), MBS
(3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS
(3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SIAB
(N-Succinimidyl(4-iodoacetyl) aminobenzoate), SULFO SIAB
(N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC
(Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate),
SULFO SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate), NHS LC SPDP
(Succinimidyl-643-(2-pyridyldithio) propionamido) hexanoate), SULFO
NHS LC SPDP (Sulfosuccinimidyl-6-[3-(2-pyridyldithio) propionamido)
hexanoate), SPDP (N-Succinimdyl-3-(2-pyridyldithio) propionate),
NHS BROMOACETATE (N-Hydroxysuccinimidylbromoacetate), NHS
IODOACETATE (N-Hydroxysuccinimidyliodoacetate), MPBH
(4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCH
(4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid hydrazide
hydrochloride), MBH (m-Maleimidobenzoic acid
hydrazidehydrochloride), SULFO EMCS
(N-(epsilon-Maleimidocaproyloxy) sulfosuccinimide), EMCS
(N-(epsilon-Maleimidocaproyloxy) succinimide), PMPI
(N-(p-Maleimidophenyl) isocyanate), KMUH
(N-(kappa-Maleimidoundecanoic acid) hydrazide), LC SMCC
(Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6-amidocaproate-
)), SULFO GMBS (N-(gamma-Maleimidobutryloxy) sulfosuccinimide
ester), SMPH
(Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO KMUS
(N-(kappa-Maleimidoundecanoyloxy) sulfosuccinimide ester), GMBS
(N-(gamma-Maleimidobutyrloxy) succinimide), DMP
(Dimethylpimelimidate hydrochloride), DMS (Dimethylsuberimidate
hydrochloride), MHBH (Wood's Reagent; Methyl-p-hydroxybenzimidate
hydrochloride, 98%), DMA (Dimethyladipimidate hydrochloride).
[0190] Components of the disclosed compositions, such as CAQK
peptides, cargo compositions, cargo molecules, surface molecules,
etc., can also be coupled using, for example, maleimide coupling.
By way of illustration, components can be coupled to lipids by
coupling to, for example,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene
glycol).sub.2000; DSPE-PEG.sub.2000-maleimide] (Avanti Polar
Lipids) by making use of a free cysteine sulfhydryl group on the
component. The reaction can be performed, for example, in aqueous
solution at room temperature for 4 hours. This coupling chemistry
can be used to couple components of co-compositions and cargo
compositions.
[0191] Components of the disclosed compositions, such as CAQK
peptides, cargo compositions, cargo molecules, surface molecules,
etc., can also be coupled using, for example, amino
group-functionalized dextran chemistry. Particles, such as, for
example, nanoparticles, nanoworms, and micelles, can be coated with
amino group functionalized dextran. Attachment of PEG to aminated
particles increases the circulation time, presumably by reducing
the binding of plasma proteins involved in opsonization (Moghimi et
al., Pharm. Rev. 53, 283-318 (2001)). The particles can have
surface modifications, for example, for reticuloendothelial system
avoidance (PEG) and homing (CAQK peptides), endosome escape
(pH-sensitive peptide; for example, Pirollo et al., Cancer Res.67,
2938-43 (2007)), a detectable agent (cargo molecule), a therapeutic
compound (cargo molecule), or a combination. To accommodate all
these functions on one particle, optimization studies can be
conducted to determine what proportion of the available linking
sites at the surface of the particles any one of these elements
should occupy to give the best combination of targeting and payload
delivery.
[0192] The disclosed peptides can have additional N-terminal,
C-terminal, or intermediate amino acid sequences, e.g., amino acid
linkers or tags. The term "amino acid linker" refers to an amino
acid sequences or insertions that can be used to connect or
separate two distinct peptides, polypeptides, or polypeptide
fragments, where the linker does not otherwise contribute to the
essential function of the composition. The term "amino acid tag"
refers to a distinct amino acid sequence that can be used to detect
or purify the disclosed peptide, where the tag does not otherwise
contribute to the essential function of the composition. The
disclosed peptides can further have deleted N-terminal, C-terminal
or intermediate amino acids that do not contribute to the essential
activity of the peptides.
[0193] Components can be directly or indirectly covalently bound to
surface molecules or each other by any functional group (e.g.,
amine, carbonyl, carboxyl, aldehyde, alcohol). For example, one or
more amine, alcohol or thiol groups on the components can be
reacted directly with isothiocyanate, acyl azide,
N-hydroxysuccinimide ester, aldehyde, epoxide, anhydride, lactone,
or other functional groups incorporated onto the surface molecules
or other components. Schiff bases formed between the amine groups
on the components and aldehyde groups on the surface molecule or
other components can be reduced with agents such as sodium
cyanoborohydride to form hydrolytically stable amine links
(Ferreira et al., J. Molecular Catalysis B: Enzymatic 2003, 21,
189-199). Components can be coupled to surface molecules and other
components by, for example, the use of a heterobifunctional silane
linker reagent, or by other reactions that activate functional
groups on either the surface molecule or the components.
[0194] Useful modes for linking components to surface molecules and
to other components include heterobifunctional linkers or spacers.
Such linkers can have both terminal amine and thiol reactive
functional groups for reacting amines on components with sulfhydryl
groups, thereby coupling the components in an oriented way. These
linkers can contain a variable number of atoms. Examples of such
linkers include, but are not limited to, N-Succinimidyl
3-(2-pyridyldithio)propionate (SPDP, 3- and 7-atom spacer),
long-chain- SPDP (12-atom spacer),
(Succinimidyloxycarbonyl-a-methyl-2-(2-pyridyldithio) toluene)
(SMPT, 8-atom spacer),
Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC,
11-atom spacer) and
Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
(sulfo-SMCC, 11-atom spacer), m-Maleimidobenzoyl-N
hydroxysuccinimide ester (MBS, 9-atom spacer),
N-(g-maleimidobutyryloxy)succinimide ester (GMBS, 8-atom spacer),
N-(g-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMBS,
8-atom spacer), Succinimidyl 6-((iodoacetyl) amino) hexanoate
(SIAX, 9-atom spacer), Succinimidyl
6-(6-(((4-iodoacetyl)amino)hexanoyl)amino)hexanoate (SIAXX, 16-atom
spacer), and p-nitrophenyl iodoacetate (NPIA, 2-atom spacer). It is
understood that a number of other coupling agents or links, with
different number of atoms, can be used.
[0195] Hydrophilic spacer atoms can be incorporated into linkers to
increase the distance between the reactive functional groups. For
example, polyethylene glycol (PEG) can be incorporated into
sulfo-GMBS. Hydrophilic molecules such as PEG have also been shown
to decrease non-specific binding (NSB) and increase hydrophilicity
of surfaces when covalently coupled. PEG can also be used as the
primary linker material.
[0196] Free amine groups of components can also be attached to
surface molecules or other components containing reactive amine
groups via homobifunctional linkers. Linkers such as
dithiobis(succinimidylpropionate) (DSP, 8-atom spacer),
disuccinimidyl suberate (DSS, 8-atom spacer), glutaraldehyde
(4-atom spacer), Bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone
(BSOCOES, 9-atom spacer), all requiring high pH, can be used for
this purpose. Examples of homobifunctional sulfhydryl-reactive
linkers include, but are not limited to,
1,4-Di-[3'-2'-pyridyldithio)propion-amido]butane (DPDPB, 16-atom
spacer) and Bismaleimidohexane (BMH, 14-atom spacer). For example,
these homobifunctional linkers are first reacted with a thiolated
surface in aqueous solution (for example PBS, pH 7.4), and then in
a second step, the thiolated antibody or protein is joined by the
link. Homo- and heteromultifunctional linkers can also be used.
[0197] Direct binding of components to thiol, amine, or carboxylic
acid functional groups on surface molecules and other components be
used to produce compositions which exhibit viral binding (due to
increased density of components, for example), resulting in
enhanced sensitivity.
[0198] As an example, when necessary to achieve high peptide
coupling density, additional amino groups can be added to the
surface molecules (such as commercially obtained SPIO) as follows:
First, to crosslink the particles before the amination step, 3 ml
of the colloid (.about.10mgFe/ml in double-distilled water) was
added to 5ml of 5M NaOH and 2 ml of epichlorohydrin (Sigma, St.
Louis, Mo.). The mixture was agitated for 24 hours at room
temperature to promote interaction between the organic phase
(epichlorohydrin) and aqueous phase (dextran-coated particle
colloid). In order to remove excess epichlorohydrin, the reacted
mixture was dialyzed against double-distilled water for 24 hours
using a dialysis cassette (10,000 Da cutoff, Pierce, Rockford
Ill.). Amino groups were added to the surface of the particles as
follows: 0.02 ml of concentrated ammonium hydroxide (30%) was added
to lml of colloid (.about.10 mg Fe/ml). The mixture was agitated at
room temperature for 24 hours. The reacted mixture was dialyzed
against double-distilled water for 24 hours. To further rinse the
particles, the colloid was trapped on a MACS.RTM. Midi magnetic
separation column (Miltenyi Biotec, Auburn Calif.), rinsed with PBS
three times, and eluted from the column with lml PBS.
[0199] Linkers are useful for achieving useful numbers and
densities of the components (such as CAQK peptides and cargo
molecules) on surface molecules. For example, linkers of fibrous
form are useful for increasing the number of components per surface
molecule or per a given area of the surface molecule. Similarly,
linkers having a branching form are useful for increasing the
number of components per surface molecule or per a given area of
the surface molecule. Linkers can also have a branching fibrous
form.
[0200] Sufficiency of the number and composition of CAQK peptides
in the composition can be determined by assessing homing to the
target and effectively delivery of the cargo molecules in a
non-human animal. The composition can include a sufficient number
and composition of CAQK peptides such that the composition homes to
the target and effectively delivers the cargo molecules. In one
example, sufficiency of the number and composition of modified
and/or unmodified CAQK peptides can be determined by assessing
cargo delivery and/or therapeutic effect on the target.
[0201] The composition can include a sufficient density and
composition of CAQK peptides such that the composition homes to the
target and effectively delivers the cargo molecules. Sufficiency of
the density and composition of CAQK peptides can be determined by
assessing cargo delivery and/or therapeutic effect on the target in
a non-human animal.
[0202] As used herein, reference to components (such as a peptide
and a cargo composition) as being "not covalently coupled" means
that the components are not connected via covalent bonds (for
example, that the peptide and a cargo composition are not connected
via covalent bonds). That is, there is no continuous chain of
covalent bonds between, for example, the peptide and a cargo
composition. Conversely, reference to components (such as a peptide
and a cargo composition) as being "covalently coupled" means that
the components are connected via covalent bonds (for example, that
the peptide and a cargo composition are connected via covalent
bonds). That is, there is a continuous chain of covalent bonds
between, for example, the peptide and a cargo composition.
Components can be covalently coupled either directly or indirectly.
Direct covalent coupling refers to the presence of a covalent bond
between atoms of each of the components. Indirect covalent coupling
refers to the absence of a covalent bond between atoms of each of
the components. That is, some other atom or atoms not belonging to
either of the coupled components intervenes between atoms of the
components. Both direct and indirect covalent coupling involve a
continuous chain of covalent bonds.
[0203] Non-covalent association refers to association of components
via non-covalent bonds and interactions. A non-covalent association
can be either direct or indirect. A direct non-covalent association
refers to a non-covalent bond involving atoms that are each
respectively connected via a chain of covalent bonds to the
components. Thus, in a direct non-covalent association, there is no
other molecule intervening between the associated components. An
indirect non-covalent association refers to any chain of molecules
and bonds linking the components where the components are not
covalently coupled (that is, there is a least one separate molecule
other than the components intervening between the components via
non-covalent bonds).
[0204] Reference to components (such as a peptide and a cargo
composition) as not being "non-covalently associated" means that
there is no direct or indirect non-covalent association between the
components. That is, for example, no atom covalently coupled to a
peptide is involved in a non-covalent bond with an atom covalently
coupled to a cargo composition. Within this meaning, a peptide and
a cargo composition can be together in a composition where they are
indirectly associated via multiple intervening non-covalent bonds
while not being non-covalently associated as that term is defined
herein. For example, a peptide and a cargo composition can be mixed
together in a carrier where they are not directly non-covalently
associated. A peptide and a cargo composition that are referred to
as not indirectly non-covalently associated cannot be mixed
together in a continuous composition. Reference to components (such
as a peptide and a cargo composition) as not being "directly
non-covalently associated" means that there is no direct
non-covalent association between the components (an indirect
non-covalent association may be present). Reference to components
(such as a peptide and a cargo composition) as not being
"indirectly non-covalently associated" means that there is no
direct or indirect non-covalent association between the
components.
[0205] It is understood that components can be non-covalently
associated via multiple chains and paths including both direct and
indirect non-covalent associations. For the purposes of these
definitions, the presence a single direct non-covalent association
makes the association a direct non-covalent association even if
there are also indirect non-covalent associations present.
Similarly, the presence of a covalent connection between components
means the components are covalently coupled even if there are also
non-covalent associations present. It is also understood that
covalently coupled components that happened to lack any
non-covalent association with each other are not considered to fall
under the definition of components that are not non-covalently
associated.
[0206] Additional homing molecules that target sites of nervous
system injury can be identified. Such homing molecules are useful
and useable in all the ways that CAQK peptides useful and useable.
The method can involve bringing into contact a test compound and
versican, tenascin-R, Hapln, or combinations thereof, and assessing
whether the test compound specifically binds to the versican,
tenascin-R, Hapln, or combination thereof. The test compound is
identified as a compound that target sites of nervous system injury
if the test compound specifically binds to the versican,
tenascin-R, Hapln, or combination thereof. In some forms, the
method can involve bringing into contact the homing molecule and
CSPG-rich extracellular matrix complexes, and assessing whether the
homing molecule specifically binds to the CSPG-rich extracellular
matrix complexes. The homing molecule is identified if the homing
molecule specifically binds to the CSPG-rich extracellular matrix
complexes. In some forms the CSPG-rich extracellular matrix
complexes can be matrix produced by U251 astrocytoma cells.
[0207] In some forms of the method, the versican, tenascin-R,
Hapln, or combination thereof can be part of extracellular matrix.
In some forms of the method, the extracellular matrix can be in or
obtained from glial scar. In some forms of the method, the
extracellular matrix is a CSPG-rich extracellular matrix complex
(extracellular matrix containing hyaluronic acid, versican,
tenascin-R, and Hapln and exemplified by the matrix of cultured
U251 astrocytoma cells). In some forms of the method, the
extracellular matrix is matrix from cultured U251 astrocytoma
cells.
[0208] In some forms of the method, assessing whether the test
compound specifically binds the versican, tenascin-R, Hapln, or
combination thereof, can be accomplished by assessing whether the
test compound specifically binds the CSPG-rich extracellular matrix
complex. In some forms of the method, the CSPG-rich extracellular
matrix complex can be at a site of nervous system injury in test
animal, where bringing into contact is accomplished by
administering the test compound to the animal intravenously.
[0209] In some forms of the method, the versican, tenascin-R,
Hapln, or combination thereof is not comprised in extracellular
matrix. In some forms of the method, the versican, tenascin-R,
Hapln, or combination thereof can be made from individual versican,
tenascin-R, and Hapln proteins.
[0210] In some forms of the method, the test compound can be
coupled to a label, wherein assessing whether the test compound
specifically binds is accomplished by detecting the label.
B. Methods of Targeting and Treating
[0211] The disclosed peptides and compositions are useful for
selective targeting nervous system injury, such as brain injury and
stroke injury, sites of glial scar formation, sites where
hyaluronic acid, versican, tenascin-R, and Hapln are being
deposited, and CSPG-rich extracellular matrix complexes. For
example, the disclosed peptides and compositions are useful for
selectively targeting acute nervous system injury, such as
traumatic brain injury and stroke injury. Thus, methods for
selectively targeting a cargo to a site of acute nervous system
injury in a subject are disclosed.
[0212] In some forms, the method involves administering the
disclosed composition to a subject having an acute nervous system
injury. The composition selectively homes to a site of the nervous
system injury in the subject thereby selectively targeting the
cargo composition of the composition to the site of the nervous
system injury.
[0213] In some forms, the nervous system injury includes a brain
injury. In some forms, the peptide selectively homes to a site of
the brain injury. In some forms, the brain injury includes
traumatic brain injury, stroke injury, or both. In some forms, the
peptide specifically binds to one or more of versican, tenascin-R,
and Hapln. In some forms, the peptide selectively homes to a site
of glial scar formation. In some forms, the peptide selectively
homes to a site where hyaluronic acid, versican, tenascin-R, and
Hapln are being deposited. In some forms, the peptide selectively
homes to CSPG-rich extracellular matrix complex.
[0214] The disclosed peptides and compositions are particularly
useful for targeting acute nervous system injury. Thus, in some
forms, the composition near the time of the injury or during the
acute phase of the injury. In some forms, the composition is
administered within 10 days of the onset of the nervous system
injury. In some forms, the composition is administered within 5
days of the onset of the nervous system injury. In some forms, the
composition is administered within 24 hours of the onset of the
nervous system injury.
[0215] Disclosed in particular are methods of selectively targeting
a cargo composition to a site of acute nervous system injury in a
subject, where the method involves administering the composition to
a subject having an acute nervous system injury, where the
composition includes a peptide and a cargo composition, where the
peptide consists of the amino acid sequence CAQK (SEQ ID NO:4),
where the cargo composition includes a surface molecule and cargo
molecule, where the surface molecule includes a nanoparticle, where
the cargo molecule is encapsulated in the nanoparticle, where the
cargo molecule is a therapeutic agent, and where the therapeutic
agent includes a functional nucleic acid. The composition
selectively homes to a site of the nervous system injury in the
subject thereby selectively targeting the cargo composition of the
composition to the site of the nervous system injury.
[0216] Studies have revealed extensive molecular heterogeneity in
the molecular targets accessible to circulating blood in different
normal tissues. In addition, pathological lesions, such as tumors
and locations of injury, impose their own changes on the accessible
molecular targets. Targeting accessible molecular targets enable
docking-based (`synaphic`) targeting to selectively deliver
diagnostics and therapeutics into a specific tissue. This approach
can produce greater efficacy and diminished side effects. The
targeted delivery principle has been established, particularly in
cancer. It has been realized that it may be more effective to
target the delivery to molecular targets accessible to circulating
blood because of their accessibility. While penetration outside of
the vasculature has been a problem for some such targeted
diagnostics and therapeutics, the presence of injury where the
targets of CAQK peptides occur provides entry points and access for
the disclosed targeted peptides and compositions.
[0217] Binding in the context of a homing molecule recognizing
and/or binding to its target can refer to both covalent and
non-covalent binding, for example where a homing molecule can bind,
attach or otherwise couple to its target by covalent and/or
non-covalent binding. Binding can be either high affinity or low
affinity, preferably high affinity. Examples of binding forces that
can be useful include, but are not limited to, covalent bonds,
dipole interactions, electrostatic forces, hydrogen bonds,
hydrophobic interactions, ionic bonds, and/or van der Waals forces.
This binding can occur in addition to that binding which occurs
with the disclosed targeted peptides and compositions.
[0218] Also disclosed more generally are methods of selectively
targeting CSPG-rich extracellular matrix complexes (extracellular
matrix containing hyaluronic acid, versican, tenascin-R, and Hapln
and exemplified by the matrix of cultured U251 astrocytoma cells).
The method can involve administering a composition to a subject,
where the composition comprises a homing molecule and a
pharmaceutically acceptable carrier. The composition selectively
homes to the CSPG-rich extracellular matrix complex thereby
selectively targeting the CSPG-rich extracellular matrix
complex.
[0219] In some forms of the method, the homing molecule can
specifically bind to one or more of versican, tenascin-R, and
Hapln. In some forms of the method, the homing molecule can
selectively home to a site where hyaluronic acid, versican,
tenascin-R, and Hapln are being deposited. In some forms of the
method, the composition can be administered intravenously. In some
forms of the method, the composition can be administered
systemically.
[0220] In some forms of the method, the composition can further
comprise a cargo composition. In some forms of the method, the
cargo composition can include one or more cargo molecules. In some
forms of the method, the cargo molecules can each independently be
a therapeutic agent, a therapeutic protein, a therapeutic compound,
a therapeutic composition, a polypeptide, a nucleic acid molecule,
a small molecule, a label, a labeling agent, a contrast agent, an
imaging agent, a fluorophore, fluorescein, rhodamine, a
radionuclide, indium-111, technetium-99, carbon-11, or carbon-13,
or combinations thereof. In some forms of the method, at least one
of the cargo molecules comprises a therapeutic agent. In some forms
of the method, at least one of the cargo molecules comprises a
functional nucleic acid. In some forms of the method, at least one
of the cargo molecules comprises a detectable agent.
[0221] 1. Targets and Subjects
[0222] The disclosed compositions are targeted and can home to
sites of nervous system injury. As used herein, "nervous system
injury" refers to an injury or damage to any part of the nervous
system. Different parts of the nervous system can be injured, which
injuries can be referred to by the part of the nervous system that
is injured. Thus, for example, central nervous system injury,
peripheral nervous system injury, brain injury, spinal cord injury,
neural injury, and neuronal injury refer to injury of the central
nervous system, peripheral nervous system, brain, spinal cord,
nerves, and neurons, respectively. Some nervous system injuries can
be distributed, such as neurodegenerative diseases (such as
Alzheimer's disease and Parkinson's disease), demyelinating
diseases (multiple sclerosis is an example), and autoimmune
diseases that affect nerves or parts or components of the nervous
system. Nervous system injury can be acute or chronic.
[0223] A nervous system injury can be from numerous causes,
including accident, disease, and condition. The disclosed peptides,
compositions, and methods are most useful for acute nervous system
injuries, which can occur from the same causes. As used herein, an
acute injury refers to an injury that occurs from a sudden event or
change in condition. For example, a traffic accident, gunshot,
fall, stroke, and heart attack can all be the cause of an acute
injury, including an acute nervous system injury. As an example,
acute brain injury can be caused by, for example, an open head
injury, a closed head injury, a deceleration injury, hypoxia, and
stroke.
[0224] Open head injuries can result from bullet wounds, crushing,
penetrating wounds, etc. The damage tends to be focal and to
involve penetration of the skull. Closed head injuries can result
from falls, motor vehicle crashes, etc. Damage can be both focal
and diffuse and the effects tend to be broad and diffuse. There is
no penetration of the skull in closed head injuries. Deceleration
injuries tend to cause diffuse axonal injury. Deceleration injuries
tend to occur because of differential movement of the skull and the
brain when the head is struck. This can result in direct brain
injury due to diffuse axonal shearing, contusion, and brain
swelling. Diffuse axonal shearing can occur when the brain is
slammed back and forth inside the skull it is alternately
compressed and stretched because of the gelatinous consistency. If
the impact is strong enough, axons can be stretched until they are
torn, resulting in axonal shearing. Hypoxia of brain tissue can be
caused by a reduction in oxygen in the blood. Hypoxia can be caused
by, for example, heart attacks, respiratory failure, drops in blood
pressure, and a low oxygen environment. Stroke of brain tissue can
be caused by interruption of blood flow to the brain or part of the
brain or by, for example, vascular blockage or bleeding.
[0225] Glial scar formation (gliosis) is a reactive cellular
process involving astrogliosis that occurs after injury to the
central nervous system. In the context of neurodegeneration,
formation of the glial scar has been shown to have both beneficial
and detrimental effects. Particularly, many neuro-developmental
inhibitor molecules are secreted by the cells within the scar that
prevent complete physical and functional recovery of the central
nervous system after injury or disease. On the other hand, absence
of the glial scar has been associated with impairments in the
repair of the blood brain barrier. Glial scars are composed of
several components: reactive astrocytes, microglia, endothelial
cells, fibroblasts, and basal membrane. Relative to the disclosed
peptides, compositions, and methods, the basal membrane is the most
significant because the histopathological extracellular matrix
making up the basal membrane includes the target of CAQK
peptides.
[0226] A main target of the disclosed peptides and compositions is
a CSPG-rich protein-carbohydrate extracellular matrix complex that
is that is rich in chondroitin sulfate proteoglycans (CSPGs) and is
produced in nervous system injuries. These CSPG-rich extracellular
matrix complexes (also referred to herein as CR-ECM and CSPG-rich
complexes) are the binding and homing target of the disclosed CAQK
peptides. In this regard, the CSPG-rich complexes contain the
molecules/structures to which the disclosed CAQK peptides bind. The
CSPG-rich extracellular matrix complexes include a number of
proteins and carbohydrate polymers that define the complex.
Although different ECM have common components and features, the
various forms of ECM are characterized by specific proteins and
carbohydrate polymers. The CSPG-rich extracellular matrix complexes
produced in nervous system injuries include components such as
hyaluronic acid, around which CSPGs assemble. Significant CSPGs
present in CSPG-rich extracellular matrix complexes produced in
nervous system injuries include phosphacan, neuron-glial antigen 2
(NG2), and members of the lectican family of CSPGs: aggrecan,
brevican, neurocan, and versican. Also present in CSPG-rich
extracellular matrix complexes produced in nervous system injuries
are glycoproteins such as tenascin, laminin, and fibronectin, and
proteins such as hyaluronan and proteoglycan link protein (Hapln).
A useful reference form of CSPG-rich extracellular matrix complexes
is the matrix produced by cultured U251 astrocytoma cells. Unless
the context indicates otherwise, reference to "CSPG-rich
extracellular matrix complexes," "CR-ECM," and "CSPG-rich
complexes" refer to the extracellular matrix characterized in this
paragraph and exemplified by the matrix produced by cultured U251
astrocytoma cells and should not be considered just a generic
reference to any extracellular matrix complex that is rich in or
have a high amount of CSPGs.
[0227] Hyaluronic acid (HA; also called hyaluronan) is an anionic,
nonsulfated glycosaminoglycan distributed widely throughout
connective, epithelial, and neural tissues. It is unique among
glycosaminoglycans in that it is nonsulfated, forms in the plasma
membrane instead of the Golgi, and can be very large, with its
molecular weight often reaching the millions (Fraser et al., J.
Intern. Med. 242(1): 27-33 (1997)). Hyaluronic acid is one of the
chief components of the extracellular matrix.
[0228] Versican is a large extracellular matrix proteoglycan that
is present in a variety of human tissues. It is encoded by the VCAN
gene (Iozzo et al., Genomics 14(4):845-51 (1992)). Versican is a
large chondroitin sulfate proteoglycan with an apparent molecular
mass of more than 1000 kDa. In 1989, Zimmermann and Ruoslahti
cloned and sequenced the core protein of fibroblast chondroitin
sulfate proteoglycan (Zimmermann and Ruoslahti, EMBO J. 8(10):
2975-81 (1989). They designated it versican in recognition of its
versatile modular structure. Versican belongs to the lectican
protein family, with aggrecan (abundant in cartilage), brevican and
neurocan (nervous system proteoglycans) as other members. Versican
is also known as chondroitin sulfate proteoglycan core protein 2 or
chondroitin sulfate proteoglycan 2 (CSPG2), and PG-M.
[0229] Phosphacan, one of the principal proteoglycans in the
extracellular matrix of the central nervous system, is implicated
in neuron-glia interactions associated with neuronal
differentiation and myelination. Although phosphacan occurs in the
CNS as a large CSPG (>800 kDa) (Faissner et al., J. Cell Biol.
126:783-799 (1994)); it is in fact a secreted splice variant of an
even larger transmembrane receptor protein tyrosine phosphatase
(RPTP), RPTP-.beta., also known as PTP-zeta). Hence phosphacan
corresponds to the entire extracellular part of the long
RPTP-.beta. receptor. These proteins are characterized by a
carbonic anhydrase-like (CA) domain at their extracellular N
terminus.
[0230] Tenascin-R (TNR) is an extracellular matrix protein
expressed primarily in the central nervous system. It is a member
of the tenascin (TN) gene family, which includes at least 3 genes
in mammals: TNC (or hexabrachion), TNX (TNXB), and TNR (Erickson,
Curr. Opin. Cell Biol. 5(5): 869-76 (1993)). The genes are
expressed in distinct tissues at different times during embryonic
development and are present in adult tissues.
[0231] Hyaluronan and proteoglycan link protein (Hapln) links
hyaluronan to versican (and other protetoglycans). The Hapln
involved in the target of the CAQK peptide appears to be Hapln4
(Spicer et al., J Biol Chem 278(23):21083-21093 (2003)).
[0232] Laminins are high-molecular weight (.about.400 kDa) proteins
of the extracellular matrix. They are a major component of the
basal lamina (one of the layers of the basement membrane), a
protein network foundation for most cells and organs. Laminins are
heterotrimeric proteins that contain an .alpha.-chain, a
.beta.-chain, and a y-chain, found in five, four, and three genetic
variants, respectively. The laminin molecules are named according
to their chain composition. The laminin family of glycoproteins are
an integral part of the structural scaffolding in almost every
tissue of an organism. They are secreted and incorporated into
cell-associated extracellular matrices.
[0233] Fibronectin is a high-molecular weight (.about.440 kDa)
glycoprotein of the extracellular matrix that binds to
membrane-spanning receptor proteins called integrins. Similar to
integrins, fibronectin binds extracellular matrix components such
as collagen, fibrin, and heparan sulfate proteoglycans (e.g.
syndecans). Fibronectin exists as a protein dimer, consisting of
two nearly identical monomers linked by a pair of disulfide bonds.
Insoluble cellular fibronectin is a major component of the
extracellular matrix. It is secreted by various cells, primarily
fibroblasts, as a soluble protein dimer and is then assembled into
an insoluble matrix in a complex cell-mediated process. Fibronectin
plays a major role in cell adhesion, growth, migration, and
differentiation, and it is important for processes such as wound
healing and embryonic development.
[0234] The CAQK peptides target and home to sites of nervous system
injury due to the presence of targets for binding of the CAQK
peptides. The targets are present at sites of injury where neural
extracellular matrix is being produced, in particular, where
CSPG-rich extracellular matrix complex (extracellular matrix
containing hyaluronic acid, versican, tenascin-R, and Hapln and
exemplified by the matrix of cultured U251 astrocytoma cells) is
being produced after injury. Thus, the CAQK peptides can be
targeted and will home to sites where CSPG-rich extracellular
matrix complex is being produced. Similarly, the CAQK are also
targeted and will home to sites where the particular targets of
CAQK peptides, hyaluronic acid, versican, tenascin-R, and Hapln,
are being deposited. In this context, "being deposited" refers to
new or greater amounts of these proteins, generally as components
of extracellular matrix.
[0235] The CAQK peptide can selectively home to a site of nervous
system injury. The CAQK peptide can selectively home to a site of
acute nervous system injury. The CAQK peptide can selectively home
to a site of brain injury. The CAQK peptide can selectively home to
a site of acute brain injury. The CAQK peptide can selectively home
to a site of stroke injury. The CAQK peptide can selectively home
to a site of acute stroke injury. The CAQK peptide can selectively
bind to one or more of versican, tenascin-R, and Hapln. The CAQK
peptide can selectively home to a site of glial scar formation. The
CAQK peptide can selectively home to a site where hyaluronic acid,
versican, tenascin-R, and Hapln are being deposited. The CAQK
peptide can selectively home to CSPG-rich extracellular matrix
complex. The composition can selectively home to a site of nervous
system injury. The composition can selectively home to a site of
acute nervous system injury. The composition can selectively home
to a site of brain injury. The composition can selectively home to
a site of acute brain injury. The composition can selectively home
to a site of stroke injury. The composition can selectively home to
a site of acute stroke injury. The composition can selectively bind
to one or more of versican, tenascin-R, and Hapln. The composition
can selectively home to a site of glial scar formation. The
composition can selectively home to a site where hyaluronic acid,
versican, tenascin-R, and Hapln are being deposited. The
composition can selectively home to CSPG-rich extracellular matrix
complex.
[0236] i. Traumatic Brain Injury (TBI)
[0237] TBI is a significant cause of disability and death worldwide
and can result from stroke, motor vehicle accidents, sports
injuries, blast injuries, assaults, and falls. Stroke in
particular, is a condition that results when poor blood flow to the
brain results in cell death. There are two main types of stroke.
Hemorrhagic stroke occurs spontaneously due to bleeding from an
aneurysm or a weakened blood vessel. Ischemia, which is the more
common type of stroke, occurs due to lack of blood flow to the
brain, resulting in tissue death. Inadequate blood flow can be
caused by many factors such as atherosclerosis, hypoglycemia,
tachycardia, hypotension, anemia, frostbite, tumors, tourniquet
application, premature discontinuation of any oral anticoagulant,
and sickle cell disease.
[0238] TBI causes neuronal damage, which results from both primary
and secondary injury mechanisms. Primary injury involves mechanical
impact and inertial forces at the time of trauma, causing cellular
strain and damage to neurons, axons, glia, and blood vessels as a
result of shearing, tearing, or stretching. Secondary injury can
happen in minutes, but can also evolve over days and even months
after initial traumatic insult, resulting from delayed biochemical,
metabolic, and cellular changes that are triggered by the primary
event. Physical damage compromises the blood brain barrier, leading
to infiltration of inflammatory cytokines and chemokines into the
brain parenchyma and initiating inflammation. During secondary
injury, proteases such as calpains and caspases contribute to cell
death due to necrosis or apoptosis (Loane et al., Trends Pharmacol.
Sci. 31:596-604 (2010); Schoch et al., Neurotherapeutics 9:323-337
(2012)). These secondary injury cascades are thought to be
responsible for the development of many of the neurological issues
that arise after TBI, and their delayed nature suggests that there
is a therapeutic window for pharmacological or other treatment to
prevent progressive tissue damage and improve outcome (Loane et
al., Trends Pharmacol. Sci. 31:596-604 (2010)).
[0239] ii. Glial Scarring
[0240] The glial cells of the central nervous system (CNS), which
include astrocytes, microglia, oligodendrocytes and their
precursors, supply both structural and physiological support, and
also respond to injury or disease. Damage to the CNS can lead to
glial scarring, a process that has both beneficial and detrimental
effects. Ultimately, the scar functions to reestablish the physical
and chemical integrity of the CNS and is central to the repair of
the blood brain barrier (Faulkner et al., J. Neurosci. 24:2143-2155
(2004)). However, the glial scar also prevents neuronal regrowth,
and is thus detrimental to the physical and functional recovery of
the CNS (Silver et al., Nat. Rev. Neurosci. 5:146-156 (2004)).
[0241] Reactive astrocytes are produced in a process called
astrogliosis, and are the main cellular component of the glial
scar. Astrocytes undergo morphological changes and produce
extracellular matrix, such as chondroitin sulfate proteoglycans
(CSPGs), which physically and chemically inhibit axon growth.
Strategies that inhibit astrogliosis or prevent the synthesis of,
or degrade CSPGs have been demonstrated to relieve axon growth
inhibition and improve function.
[0242] CSPGs are generally secreted from cells, and are structural
components of a variety of human tissues, including cartilage. They
are composed of a core protein and a sugar side chain. The core
protein is generally a glycoprotein, and the side chains are
glycosaminoglycan (GAG) sugar chains attached through a covalent
bond. Among CSPGs that have been identified are aggrecan (CSPG1),
versican (CSPG2), neurocan (CSPG3), CSPG4-6, brevican (CSPG7),
CSPG8, and phosphacan. Neurocan, brevican, versican, and aggrecan
all share similar N-terminal and C-terminal domains, making up the
lectican family of proteoglycans. Lecticans interact with
hyaluronan and tenascin-R to form a ternary complex. Aggrecan is a
major component of extracellular matrix in cartilage and the
function of joints, whereas Versican is widely expressed in a
number of connective tissues, including those in vascular smooth
muscle, skin epithelial cells, and the cells of central and
peripheral nervous system. The expression of neurocan and brevican
is largely restricted to neural tissues.
[0243] CSPGs play key roles in neural development and glial scar
formation. They are involved in cell processes such as cell
adhesion, cell growth, receptor binding, cell migration, and
interaction with other extracellular matrix constituents. They also
interact with laminin, fibronectin, tenascin, and collagen. CSPGs
are known to inhibit axon regeneration after spinal cord injury, by
acting as a barrier against new axons growing into the injury site.
CSPGs play a crucial role in explaining why the spinal cord doesn't
self-regenerate after an injury.
[0244] Plasticity, associated with some return of brain function in
affected areas, has been attributed to down-regulation of CSPGs.
Rats that were able to recover from induced stroke exhibited
down-regulation of several CSPGs, including aggrecan, versican, and
phosphacan (Galtrey et al., Brain Res. Rev. 54:1-18 (2007)). Rats
that did not return any brain function did not have significant
down-regulation of CSPGs. The reduction of CSPGs in rats that
returned some brain function after stroke suggests that more
neurological connections could be made with less CSPGs present.
Medications that are able to down-regulate CSPGs may help return
more brain function to stroke patients.
[0245] CSPGs have also been implicated in Alzheimer's disease and
epilepsy. Although Alzheimer's disease is mostly characterized by
neurofibrillary tangles and senile plaques, these features in the
postmortem brains of Alzheimer's patients have indicated the
presence of CSPGs as well. CSPG4 and CSPG6, both localized on the
perimeter of neurofibrillary tangles and senile plaques, were found
on dystrophic neurons as well. Given the inhibitory effects of
CSPGs, these results suggest that CSPGs play an important role in
Alzheimer's disease progression, and could be responsible for
facilitating the regression of neurons around neurofibrillary
tangles and senile plaques. Medications that target the CSPGs in
the neurofibrillary tangles and senile plaques may help to
alleviate some of the symptoms of Alzheimer's disease.
[0246] Epilepsy is a disease characterized by seizures that are
caused by excessive neurological activity in the brain. Researchers
have observed that CSPGs are somewhat removed from the brain in
epilepsy patients. Studies have shown a decrease in phosphacan in
both the temporal lobe and the hippocampus in epilepsy cases,
suggesting that there CSPGs play a role in the control of axonal
regrowth (Galtrey et al., Brain Res. Rev. 54:1-18 (2007)). In
addition, brain-derived neurotrophic factor (BDNF) mRNA and protein
have been shown to be upregulated in the hippocampus by seizure
activity in animal models. BDNF, a protein that is primarily
located in the CNS where it acts on cells in the brain and the eye,
causes certain types of nerve cells to survive and grow. In the
peripheral nervous system, BDNF promotes the growth of sensory and
motor neurons. In the brain, BDNF is released by either a nerve
cell or a support cell, such as an astrocyte, and then binds to a
receptor on a nearby nerve cell. This binding results in the
production of a signal which can be transported to the nucleus of
the receiving nerve cell. There, it prompts the increased
production of proteins associated with nerve cell survival and
function. Infusion of anti-BDNF agents or use of BDNF knockout mice
has been shown to inhibit epileptogenesis in animal models (Binder
et al., Growth Factors 22:123-131 (2004)).
[0247] In addition to the conditions described above, glial
scarring occurs in a variety of other conditions involving the CNS,
such as Korakoff's syndrome, multiple system atrophy, prion
disease, multiple sclerosis, AIDS dementia complex, vasculitis,
Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), and
Huntington's disease (McMillian et al., Trends Neurosci. 17:138-142
(1994)). In autoimmune conditions such as multiple sclerosis,
myelin damage may be exacerbated by cytokines produced by both
active astrocytes and microglia. This could alter blood brain
barrier permeability, allowing the migration of lymphocytes into
the CNS, thus amplifying the autoimmune effect (Barron, J. Neurol.
Sci. 134:57-68 (1995)). ALS has been shown to involve reactive
astrocytes through either a loss of their neuroprotective ability
or through the gain of neurotoxic effects. Late stages of ALS are
also characterized by significant astrogliosis and astrocyte
proliferation around areas of degeneration (Verkhratsky et al.,
Neurotherapeutics 7:399-412 (2010)).
[0248] 2. Treating
[0249] The disclosed peptides and compositions are useful for
treating nervous system injuries. By selectively targeting nervous
system injury, such as brain injury and stroke injury, sites of
glial scar formation, sites where hyaluronic acid, versican,
tenascin-R, and Hapln are being deposited, and CSPG-rich
extracellular matrix complexes, the disclosed compositions can
deliver therapeutic agents to the site where they can be most
effective. The composition selectively homes to a site of the
nervous system injury in the subject thereby selectively targeting
the cargo composition of the composition to the site of the nervous
system injury and allowing therapeutic agents in the cargo
composition to act at the site of the nervous system injury.
[0250] In some forms, the nervous system injury includes a brain
injury. In some forms, the peptide selectively homes to a site of
the brain injury. In some forms, the brain injury includes
traumatic brain injury, stroke injury, or both. In some forms, the
peptide specifically binds to one or more of versican, tenascin-R,
and Hapln. In some forms, the peptide selectively homes to a site
of glial scar formation. In some forms, the peptide selectively
homes to a site where hyaluronic acid, versican, tenascin-R, and
Hapln are being deposited. In some forms, the peptide selectively
homes to CSPG-rich extracellular matrix complex.
[0251] The composition can be administered by any suitable route.
In some forms, the composition is administered intravenously. In
some forms, the composition is administered systemically. In some
forms, the composition is not administered locally.
[0252] The disclosed peptides and compositions are particularly
useful for targeting acute nervous system injury. Thus, in some
forms, the composition near the time of the injury or during the
acute phase of the injury. In some forms, the composition is
administered within 10 days of the onset of the nervous system
injury. In some forms, the composition is administered within 5
days of the onset of the nervous system injury. In some forms, the
composition is administered within 24 hours of the onset of the
nervous system injury.
[0253] Disclosed in particular are methods of selectively targeting
a cargo composition to a site of acute nervous system injury in a
subject, where the method involves administering the composition to
a subject having an acute nervous system injury, where the
composition includes a peptide and a cargo composition, where the
peptide consists of the amino acid sequence CAQK (SEQ ID NO:4),
where the cargo composition includes a surface molecule and cargo
molecule, where the surface molecule includes a nanoparticle, where
the cargo molecule is encapsulated in the nanoparticle, where the
cargo molecule is a therapeutic agent, and where the therapeutic
agent includes a functional nucleic acid. The composition
selectively homes to a site of the nervous system injury in the
subject thereby selectively targeting the cargo composition of the
composition to the site of the nervous system injury.
[0254] The disclosed methods can be most effective while the target
of the CAQK peptide is present and accessible at the site of
nervous system injury, at least a number of days after the onset of
the nervous system injury. The effectiveness of different
therapeutic agents for nervous system injuries generally also
depend on their use soon after the onset of a nervous system
injury. The disclosed methods of treatment generally will be
performed as part of a suite of treatments relating to that cause
of the nervous system injury. For example, emergency treatment for
traumatic brain injuries should be started within the so-called
"golden hour" following the injury. Treatment depends on the
recovery stage of the patient. In the acute stage, the primary aim
of the medical personnel is to stabilize the patient and focus on
preventing further injury because little can be done to reverse the
initial damage caused by trauma. The main treatments for the
subacute and chronic states of recovery focus on
rehabilitation.
[0255] In one aspect, the compositions described herein can be
administered to a subject comprising a human or an animal
including, but not limited to, a mouse, dog, cat, horse, bovine or
ovine and the like, that is in need of alleviation or amelioration
from a recognized medical condition.
[0256] The dosages or amounts of the compositions, cargo
compositions, and cargo molecules are large enough to produce the
desired effect in the method by which delivery occurs. The dosage
should not be so large as to cause adverse side effects, such as
unwanted cross-reactions, anaphylactic reactions, and the like.
Generally, the dosage will vary with the age, condition, sex and
extent of the disease in the subject and can be determined by one
of skill in the art. The dosage can be adjusted by the individual
physician based on the clinical condition of the subject involved.
The dose, schedule of doses and route of administration can be
varied.
[0257] The efficacy of administration of a particular dose of the
compositions, cargo compositions, and cargo molecules according to
the methods described herein can be determined by evaluating the
particular aspects of the medical history, signs, symptoms, and
objective laboratory tests that are known to be useful in
evaluating the status of a subject suffering a nervous system
injury or other diseases and/or conditions. These signs, symptoms,
and objective laboratory tests will vary, depending upon the
particular disease or condition being treated or prevented, as will
be known to any clinician who treats such patients or a researcher
conducting experimentation in this field. For example, if, based on
a comparison with an appropriate control group and/or knowledge of
the normal progression of the disease in the general population or
the particular individual: (1) a subject's physical condition is
shown to be improved (e.g., a tumor has partially or fully
regressed), (2) the progression of the disease or condition is
shown to be stabilized, or slowed, or reversed, or (3) the need for
other medications for treating the disease or condition is lessened
or obviated, then a particular treatment regimen will be considered
efficacious.
[0258] Any of the compositions can be used therapeutically and can
include or be used in combination with a pharmaceutically
acceptable carrier. The compositions described herein can be
conveniently formulated into pharmaceutical compositions composed
of one or more of the compositions in association with a
pharmaceutically acceptable carrier. See, e.g., Remington's
Pharmaceutical Sciences, latest edition, by E. W. Martin Mack Pub.
Co., Easton, Pa., which discloses typical carriers and conventional
methods of preparing pharmaceutical compositions that can be used
in conjunction with the preparation of formulations of the
compositions described herein and which is incorporated by
reference herein. These most typically would be standard carriers
for administration of compositions to humans. In one aspect, humans
and non-humans, including solutions such as sterile water, saline,
and buffered solutions at physiological pH. Other compounds can be
administered according to standard procedures used by those skilled
in the art.
[0259] The pharmaceutical compositions described herein can
include, but are not limited to, carriers, thickeners, diluents,
buffers, preservatives, surface active agents and the like in
addition to the molecule of choice. Pharmaceutical compositions can
also include one or more active ingredients such as antimicrobial
agents, antiinflammatory agents, anesthetics, and the like.
[0260] The disclosed compositions are most useful for delivery
systemically, and particularly for intravenous administration.
However, other routes and modes of delivery are not precluded for
the disclosed compositions. The manner of delivery can vary
depending on whether local or systemic treatment is desired, and on
the area to be treated. Thus, for example, a compound or
pharmaceutical composition described herein can be administered as
an ophthalmic solution and/or ointment to the surface of the eye.
Moreover, a compound or pharmaceutical composition can be
administered to a subject vaginally, rectally, intranasally,
orally, by inhalation, or parenterally, for example, by
intradermal, subcutaneous, intramuscular, intraperitoneal,
intrarectal, intraarterial, intralymphatic, intravenous,
intrathecal and intratracheal routes. Parenteral administration, if
used, is generally characterized by injection. Injectables, the
preferred form, can be prepared in conventional forms, either as
liquid solutions or suspensions, solid forms suitable for solution
or suspension in liquid prior to injection, or as emulsions.
Another approach for parenteral administration involves use of a
slow release or sustained release system such that a constant
dosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which is
incorporated by reference herein.
[0261] In some forms, the disclosed methods can have a therapeutic
effect. In some forms, the therapeutic effect can be reducing
damage of a nervous system injury. In some forms, the therapeutic
effect can be increasing retention of nervous system function
following a nervous system injury. In some forms, the subject can
have one or more sites to be targeted, where the composition homes
to one or more of the sites to be targeted. In some forms, the
subject can have a site of nervous system injury, where the
composition has a therapeutic effect at the site of nervous system
injury.
[0262] The terms "high," "higher," "increases," "elevates," or
"elevation" refer to increases above basal levels, e.g., as
compared to a control. The terms "low," "lower," "reduces," or
"reduction" refer to decreases below basal levels, e.g., as
compared to a control.
[0263] The term "inhibit" means to reduce or decrease in activity
or expression. This can be a complete inhibition of activity or
expression, or a partial inhibition. Inhibition can be compared to
a control or to a standard level. Inhibition can be 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100%.
[0264] The term "monitoring" as used herein refers to any method in
the art by which an activity can be measured.
[0265] The term "providing" as used herein refers to any means of
adding a compound or molecule to something known in the art.
Examples of providing can include the use of pipettes, pipettemen,
syringes, needles, tubing, guns, etc. This can be manual or
automated. It can include transfection by any mean or any other
means of providing nucleic acids to dishes, cells, tissue,
cell-free systems and can be in vitro or in vivo.
[0266] The term "preventing" as used herein refers to administering
a compound prior to the onset of clinical symptoms of a disease or
conditions so as to prevent a physical manifestation of aberrations
associated with the disease or condition.
[0267] The term "in need of treatment" as used herein refers to a
judgment made by a caregiver (e.g. physician, nurse, nurse
practitioner, or individual in the case of humans; veterinarian in
the case of animals, including non-human mammals) that a subject
requires or will benefit from treatment. This judgment is made
based on a variety of factors that are in the realm of a care
giver's expertise, but that include the knowledge that the subject
is ill, or will be ill, as the result of a condition that is
treatable by the disclosed compositions.
[0268] As used herein, "subject" includes, but is not limited to,
animals, plants, bacteria, viruses, parasites and any other
organism or entity. The subject can be a vertebrate, more
specifically a mammal (e.g., a human, horse, pig, rabbit, dog,
sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a
fish, a bird or a reptile or an amphibian. The subject can be an
invertebrate, more specifically an arthropod (e.g., insects and
crustaceans). The term does not denote a particular age or sex.
Thus, adult and newborn subjects, as well as fetuses, whether male
or female, are intended to be covered. A patient refers to a
subject afflicted with a disease or disorder. The term "patient"
includes human and veterinary subjects.
[0269] By "treatment" and "treating" is meant the medical
management of a subject with the intent to cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or
disorder. This term includes active treatment, that is, treatment
directed specifically toward the improvement of a disease,
pathological condition, or disorder, and also includes causal
treatment, that is, treatment directed toward removal of the cause
of the associated disease, pathological condition, or disorder. In
addition, this term includes palliative treatment, that is,
treatment designed for the relief of symptoms rather than the
curing of the disease, pathological condition, or disorder;
preventative treatment, that is, treatment directed to minimizing
or partially or completely inhibiting the development of the
associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder. It is
understood that treatment, while intended to cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or
disorder, need not actually result in the cure, ameliorization,
stabilization or prevention. The effects of treatment can be
measured or assessed as described herein and as known in the art as
is suitable for the disease, pathological condition, or disorder
involved. Such measurements and assessments can be made in
qualitative and/or quantitiative terms. Thus, for example,
characteristics or features of a disease, pathological condition,
or disorder and/or symptoms of a disease, pathological condition,
or disorder can be reduced to any effect or to any amount.
[0270] A cell can be in vitro. Alternatively, a cell can be in vivo
and can be found in a subject. A "cell" can be a cell from any
organism including, but not limited to, a bacterium.
[0271] By the term "effective amount" of the disclosed
compositions, peptides, cargo compositions, cargo molecules,
surface molecules, etc. is meant a nontoxic but sufficient amount
of the compound to provide the desired result. The term effective
amount is generally used to refer to compounds and compositions
that are intended to have certain effects. As discussed elsewhere
herein, the exact amount required will vary from subject to
subject, depending on the species, age, and general condition of
the subject, the severity of the disease that is being treated, the
particular compound used, its mode of administration, and the like.
Thus, it is not possible to specify an exact "effective amount."
However, an appropriate effective amount can be determined by one
of ordinary skill in the art using only routine
experimentation.
[0272] By "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material can
be administered to a subject along with the selected compound
without causing any undesirable biological effects or interacting
in a deleterious manner with any of the other components of the
pharmaceutical composition in which it is contained.
EXAMPLES
Example 1
Isolation of Brain Injury Selective Peptide by Phage Display
[0273] Materials and Methods
[0274] Brain Injury Models. All animal experiments were conducted
under an approved protocol of the Institutional Animal Care and Use
Committee of Sanford Burnham Prebys Medical Discovery Institute.
Eight to ten week old male BL6 mice were anesthetized with 4%
isoflurane (Aerrane; Baxter, UK) in 70% N.sub.2O and 30% O.sub.2
and positioned in a stereotaxic frame. Using a head restraint, a
5-mm craniotomy was made using a portable drill and a trephine over
the right parietotemporal cortex and the bone flap was removed.
Penetrating brain injury model was used as described (Kielian et
al., J. Immunol. 166:4634-4643 (2001); Kielian, J.
Neuroinflammation 1:16 (2004)). Nine needle punctures using a 21G
needle were made 3 mm deep according to a 3.times.3 grid, spaced 1
mm in width and 1 mm in height. For traumatic brain injury, a
Controlled Cortical Impact (CCI) model was used as described
(Krajewska et al., PLoS One 6:e24341 (2011)). Mice were subjected
to CCI using the benchmark stereotaxic impactor (Impact One.TM.;
myNeuroLab.com) with the actuator part mounted directly on the
stereotaxic instrument. The impactor (3 mm in diameter) tip
accelerated down to the 1.0 mm distance, reaching the preset
velocity of 3 m/s, and the applied electromagnetic force remained
there for the dwell time of 85 ms, and then retracted
automatically. The contact sensor indicated the exact point of
contact for reproducible results. In both models, facemask
anesthesia (1-2% isoflurane in 70%/30% nitrous oxide/oxygen) was
used during the entire procedure and afterwards, the scalp was
closed with sutures, anesthesia discontinued, and mice were
administered buprenorphine i.p. for pain control. For the first 2
hours after injury, mice were closely monitored in their cages.
[0275] In vivo Phage Display. Six hours after brain injury, mice
were intravenously injected with le10 pfu of a CX7C naive phage
library, in 100 .mu.L, of PBS. The library was allowed to circulate
for 30 minutes, after which mice were anesthetized with 2.5%
avertin and perfused with PBS intracardially. Brains were
extracted, and the tissue surrounding the injury and the
corresponding region from the contralateral side was isolated.
Tissues were homogenized in LB-NP 40 (1%) and phage was processed
as described (Teesalu et al., Methods Enzymol 503:35-56 (2012)).
Briefly, recovered phages were titered and amplified in E. coli
BLT5403 and purified for input for second round of screening. The
colonies recovered from second round were sequenced using Sanger
sequencing (Eton biosciences, San Diego, USA). Alternatively, after
first round, the phages in the lysate were rescued by amplification
in E. coli and peptide-encoding portion of the phage genome was
sequenced using Ion Torrent high throughput sequencing.
[0276] Peptide Synthesis and Coupling. The peptides were
synthesized on a microwave-assisted automated peptide synthesizer
(Liberty; CEM, Matthews, N.C.) following Fmoc/t-Bu (Fmoc:Fluorenyl
methoxy carbonyl, t-Bu: tertiary-butyl) strategy on rink amide
resin with HBTU
(N,N,N',N'-Tetramethyl-O-(1H-benzotriazol-1-yl)uranium
hexafluorophosphate (OR)
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate) activator, collidine activator base and 5%
piperazine for deprotection. Fluorescein and biotin tags were
incorporated during synthesis at the N-terminus of the sequence.
Cleavage using a 95% TFA Trifluoro acetic acid followed by
purification gave peptides with >90% purity. Peptides were
lyophilized and stored at -20.degree. C.
[0277] Animal Experiments for Skin and Liver Injury. For liver
injury model, animals were anesthetized and a midline laparotomy
was performed by first cutting the skin, bluntly separating the
muscle, and then lifting the peritoneum with sterile forceps. A
small hole was made into the lifted peritoneum and the hole was
carefully expanded (without damage to the internal organs) in both
directions along the midline. Once good exposure to liver was
obtained, the artery was clamped (Roboz surgical (RS-5420)) and an
excision wound 2 mm in depth on the surface of one of the liver
lobes was made. The clamp was removed from the artery allowing
blood to flow. The incisions on peritoneum, muscle and skin were
closed. The mice were then placed on a heating pad in their cage
and monitored closely until they recovered from anesthesia.
[0278] For skin wounds, induction of anesthesia was done and the
skin was cleaned with alcohol and betadine and four 6 or 8 mm skin
biopsies were made to the back skin of the mouse. None of the skin
wounds were covered or sutured closed in order to guarantee optimal
and infection-free healing. To test peptide homing, FAM-labeled
peptide (50 nmoles) was injected i.v. 6 hours after injury and
allowed to circulate for 30 minutes. Mice were perfused
intracardially with saline and organs were isolated and analyzed by
immunostaining.
[0279] CLARITY Imaging of Brain. CLARITY was performed on
freshly-extracted brain tissues as described (Chung et al., Nature
497:332-337 (2013)). Briefly, mice were intravenously injected with
FAM-labeled peptides 6 hours after brain injury. After 30-minute
circulation, mice were intracardially perfused with PBS and
hydrogel solution (Acrylamide (4%), bis (0.05%), VA-044 Initiator
(0.25%), paraformaldehyde 4% in PBS). Following perfusion, the
tissues were incubated in the hydrogel solution at 4.degree. C. for
2-3 days. At UCSD Neuroscience Core and Light Microscopy Facility
the tissues were then degased with nitrogen and incubated at
37.degree. C. for 3-4 hours for polymerization. Samples were then
passively cleared in 4% SDS solution for around 4 weeks until the
tissue became transparent. Finally, the samples were washed in
PBS-T for 2 days and incubated in gradient glycerol solutions: 30,
50 and 80%, about 1 day each and stored in 80% glycerol at room
temperature until imaged on a confocal microscope (Leica SP5).
[0280] Results
[0281] To isolate peptides that specifically target brain injury,
unilateral puncturing stab wound injuries were inflicted to the
right hemisphere of adult male mice (FIG. 1A). The penetrating
brain injury (PBI) resulted in rupturing of BBB visualized by
selective leakage of mouse IgG into the brain parenchyma on the
injured side. PBI also caused cortical tissue loss, axonal damage,
and loss of myelin in the corpus callosum, and was accompanied by
an increase in glysocaminoglycan deposition in the injured
hemisphere.
[0282] A T7 phage library that displays on the phage surface
9-amino acid cyclic peptides with the general composition of CX7C
(SEQ ID NO:3) (C=cysteine; X=any amino acid) (Teesalu et al.,
Methods Enzymol 503:35-56 (2012)) was intravenously injected 6
hours after PBI. Phage was harvested 30 min after injection from
the injury site and the corresponding contralateral hemisphere.
Phage recovery was 10-fold higher from the injured hemisphere than
from the uninjured contralateral side, indicating BBB breakdown
caused by the injury. High-throughput sequencing analysis of the
recovered phage pool revealed a striking enrichment of phage with
the tetra-peptide insert, CAQK (SEQ ID NO:4), which comprised 22%
(1.28.times.10.sup.5 pfu) of total recovered phage pool
(6.4.times.10.sup.5 pfu). In addition to the truncated CAQK
peptide, full-length (9aa) cyclic inserts starting with the CAQK
motif were also recovered at lower frequency. A second round of
biopanning increased the CAQK fraction to 83% of the total
recovered phage pool. Interestingly, there was some CAQK phage
recovery from the contralateral side (4%), which suggested a mild
impairment triggered through the contralateral injury (FIG. 1B). No
CAQK was recovered from the brain of a normal mouse injected with
the phage library.
[0283] To validate the selectivity of CAQK, a fluoresceinamine
(FAM)-labeled CAQK peptide was chemically synthesized. In agreement
with the phage screening results, the synthetic FAM-CAQK peptide
(SEQ ID NO:5), when injected intravenously, showed selective homing
to the injured brain upon macroscopic examination (FIG. 1C), and
immuno-histochemical staining for the FAM-label on the peptide. A
FAM-labeled control peptide (CGGK) (SEQ ID NO:7) of the same length
and overall charge as CAQK yielded minimal fluorescent signal. No
CAQK accumulation was observed in healthy brain or in other major
tissues after 30-minute circulation, except in the kidney, which is
the common route for peptide elimination from the circulation (FIG.
1D).
[0284] To investigate if CAQK targets other types of brain
injuries, peptide homing was tested in a controlled cortical impact
injury (CCI) model. Without penetrating injury, this model mimics
cortical tissue loss, axonal injury, concussion and BBB dysfunction
of TBI (Xiong et al., Nature Reviews Neuroscience 14:128-142
(2013)). CAQK homed to the injured area in the brain in this model.
Binding of CAQK peptide was specific to brain injuries as no
peptide accumulation was detected in perforating injuries inflicted
on the liver and skin (FIG. 1D). These findings suggest that the
binding epitope for CAQK peptide is specific for sites of brain
injury, at least in the models tested.
[0285] CAQK homing to PBI was observed up to 5 days after the
injury (FIG. 1E), indicating a potential window for effective
systemic CAQK-targeting. To visualize peptide accumulation across
the entire brain, whole brains were processed and made transparent
using the CLARITY protocol (Chung at al., Nature 497:332-337
(2013)) and FAM-CAQK was visualized in the transparent tissue.
FAM-CAQK signal in the clarified brain was restricted to the
injured quadrant of the brain and was higher than the signal from
FAM-CGGK control. CAQK accumulated in the region of the commissural
fibers of the corpus callosum and in ring-shaped cellular
structures in the cortex. CAQK accumulation led to a greater
retention in the injured brain, as the peptide signal was visible 3
hours after injection whereas the control peptide was completely
washed out.
Example 2
CAQK Homing is Specific to Sites of Brain Injury
[0286] Materials and Methods
[0287] Silver Nanoparticles Synthesis and Targeting. Silver
nanoparticles (AgNPs) with PEG coating and peptide functionality
were prepared as reported previously with some modifications (Braun
et al., Nature Materials 13:904-911 (2014)). AgNPs of .about.20 nm
diameter were synthesized by tannic acid reduction of silver
nitrate in citrate solution (Dadosh, Mater Lett 63:2236-2238
(2009)). AgNO.sub.3 (252 mg) dissolved in 2.5 L water was stirred
and heated to 60.degree. C., then 50 mL water containing tannic
acid (6.1 mg) and trisodium citrate dihydrate (1 g) was added.
After 3 min the solution was brought to a boil for 20 min. Final
optical density at 400 nm was .about.10. Lipoic PEG amine (LPN,
51.9 mg, 3400 g/mol, Nanocs) was reduced in 84 mM
tris-carboxylethyl phosphine (TCEP pH 7.0, Sigma) in 4.1 mL water
for 3 h. AgNPs were portioned to 500 mL and heated to 50.degree.
C., then LPN solution (0.79 mL) was added, followed by 0.25 mL 0.5
M TCEP. After 30 min, the solution was cooled to room temperature
(RT). Tween 20 (T20, 0.25 mL, 10% in water) and 20 mL 2 M NaCl were
added, and incubated overnight at 4.degree. C. AgNPs were
concentrated 50-fold and purified by stirred cell (Millipore) with
a 100 kDa membrane into 0.5.times. PBS with 0.005% T20 and 5 mM
TCEP, then passivated with 0.03 mM N-acetyl-L-cysteine methyl ester
(Sigma), and 0.10 mM tetracysteine peptide (acetyl-CCPGCC-amide,
LifeTein) (SEQ ID NO:8), washed at 20 kRCF and resuspended at 300
O.D. in 0.05 M phosphate buffer with 0.005% T20. This product could
be stored at least 6 months at 4.degree. C. A bifunctional linker
was reacted with the amine to introduce maleimide groups
(NHS-PEG-Mal, 5 kDa JenKem USA), washed by centrifugation, and
reacted with cysteine peptide (FAM-x-CAQK-NH2) (SEQ ID NOP:9) or a
control thiol-containing peptide, or L-cysteine (Sigma). The
product was washed in PBS with 0.005% T20 (PBST), filtered (0.22
.mu.m), with a typical final optical density of 150 at the Ag
plasmon peak of 400 nm. This concentration was estimated to be
.about.30 nM in AgNPs using an extinction coefficient of 5.times.10
9 M.sup.-1 cm.sup.-1 for spherical silver obtained from Braun et
al., Nature Materials 13:904-911 (2014) (Navarro et al., The
Analyst 138:583-592 (2013)). Fixed tissue sections were stained for
Ag using Silver Enhance (Thermo Fischer), counterstained with
Nuclear Fast Red (Sigma), and mounted in DPX (Sigma). Silver
nanoparticle signal in tissue sections was quantified by Image J
software by isolating grey pixels that represent Ag. For animal
experiments, 35 nM of peptide-conjugated silver nanoparticles were
injected intravenously in mice 6 hours after PBI. The particles
were allowed to circulate for 2 hours, the mice were perfused and
the brains were isolated. Silver accumulation in the brain was
analyzed by silver staining autometallography with counterstaining
with nuclear fast red (Sigma).
[0288] Homing Studies and Tissue Sections. Animals were
intravenously injected, six hours after injury, with 50 nmoles of
peptide dissolved in PBS, and allowed to circulate for 30 minutes.
Mice were perfused intracardially with saline and organs were
isolated and imaged using the Illumatool Bright Light System
LT-9900 (Lightools Research). Brains and organs were placed in 4%
PFA at pH 7.4 overnight, washed with PBS and placed in graded
sucrose solutions overnight before OCT embedding. 10 .mu.m thick
sections were cut and analyzed by immunofluorescence. For a
complete histological analysis, sections were stained with the
Movat pentachrome kit (American Mastertech Inc.) following the
manufacturer's instructions.
[0289] Tissue Section Overlay of Silver Nanoparticles. Overlay
experiments to analyze ex vivo binding were carried out on frozen
brain tissue sections following the same protocol for
immunofluorescence staining described above taking the
nanoparticles as if they were the primary antibody and using no
secondary antibody. Peptide coated silver nanoparticles in PBS-T at
a 1 nM concentration were used and the incubation time was 1 hour
at 37.degree. C. Sections were imaged with fluorescent microscopy
by looking at intrinsic emission from the FAM tag on the
peptide.
[0290] Results
[0291] To further characterize the binding of CAQK to the injured
brain area, overlay binding experiments with silver nanoparticles
conjugated with CAQK (CAQK-NPs) on mouse brain sections were
carried out. CAQK-NPs showed strong binding to the injured brain
sections, whereas the binding of control NPs (CGGK-NP) was
negligible. Low binding of CAQK-NPs to brain sections from normal
animals was observed, suggesting the presence of low levels of the
peptide binding epitope in normal brain, and its elevation upon
injury. Similar binding pattern of CAQK-NPs was also observed in
the controlled cortical impact model. Binding specificity was
confirmed by inhibiting the CAQK-NP binding with excess of free
CAQK, which resulted in near complete inhibition.
Example 3
CAQK Peptide Interacts with Component of the Brain ECM
[0292] Materials and Methods
[0293] Affinity Chromatography and Proteomics. For identifying CAQK
binding proteins, mouse brains with brain injury were collected 6
hrs after injury. Using liquid nitrogen, the brains were crushed
and ground into powder using a mortar and pestle. Next, brain
tissue was lysed in PBS containing 200 mM
n-octyl-beta-D-glucopyranoside and protease inhibitor cocktail
(Roche) as described (Teesalu et al., PNAS 16157-16162 (2009)). The
cleared lysate was loaded on to CAQK or control peptide (CGGK)
coated Sulfolink-agarose beads (Pierce, Waltham, Mass.) and
incubated at 4.degree. C. for 3-4 hrs. The column was washed with
wash buffer (75 mM octyl-beta-D-glucopyranoside and protease
inhibitor cocktail in PBS) followed by washing with 0.5 mM control
peptide in wash buffer to remove nonspecifically bound proteins.
The bound proteins were eluted with 2 mM free CAQK peptide. The
eluted factions were pooled, their protein concentration determined
by using bicinchoninic acid (BCA) protein assay (Thermo Fischer)
and the samples were digested using the Filter-aided Sample
Preparation (FASP) method (Wisniewski et al., Nat Methods 6:359-362
(2009)). Finally, the digested samples were desalted, dried, and
subjected to LC-MS/MS analysis at the Proteomics Core facility of
the Sanford Burnham Prebys Medical Discovery Institute. All mass
spectra were analyzed with MaxQuant software version 1.5.0.25. The
MS/MS spectra were searched against the Mus musculus Uniprot
protein sequence database (version July 2014). For the data in
Table 1, proteins belonging to the PNN complex were identified by
peptide-affinity chromatography and mass spectrometry analysis on
mouse PBI brains. The LFQ intensities were derived using MAXQuant
software and averaged for three technical replicates. Intensities
are plotted on a log.sub.2 scale. Empty column denotes protein was
not detected.
[0294] Immunofluorescence. Frozen sections were permeabilized using
PBS-Triton (0.2%), blocking was carried out using 5% blocking
buffer: 5% BSA, 1% goat serum, 1% donkey serum in PBS-T. Primary
antibodies were incubated in diluted (1%) blocking buffer overnight
at dilutions 1/100 or 1/200 at 4.degree. C., washed with PBS-T and
incubated with secondary antibodies diluted 1/200 or 1/500 in 1%
diluted buffer for one hour at room temperature, subsequently
washed with PBS-T, counterstained with DAPI 1 .mu.g/mL in PBS for
five minutes, washed with PBS, mounted using mounting media (Vector
Biolabs), and imaged on a confocal microscope (Zeiss LSM-710).
Staining was done using the following antibodies and reagents:
Fluorescein (Invitrogen A889), Versican (abcam, ab177480), Hapln4
(R&D systems, AF4085), tenascin R (R&D systems, AF3865),
WFA (Sigma, L1516), CSPG (Sigma, C8035), NG2 and olig2 (gift from
Dr. William Stallcup at SBPMDI), GFAP (Dako, Z0334) and MBP
(Millipore -MAB386).
[0295] Phage Binding to ECM. Cells grown as confluent monolayer in
a 96-well plate were gently removed by an enzyme free cell
dissociation buffer (Thermo Fisher Scientific) and plates blocked
with 200 .mu.L of 0.5% bovine serum albumin (BSA) in PBST for 1 h
at 37.degree. C. Phage was incubated in the plate at 4.degree. C.
for overnight and unbound phage was removed by washing 3 times with
200 .mu.L of PB ST. The bound phage was detected by incubating with
an in-house generated anti-T7 phage antibody for 1 hour at
4.degree. C. Following washing, horseradish peroxidase
(HRP)-labeled anti-rabbit IgG (Sigma-Aldrich) was diluted 1:1000 in
PBS, and 100 .mu.L was added to the wells, followed by 30 minute
incubation at room temperature and washing 3 times. Next, 100 .mu.L
of OPD silver and gold substrate (Sigma-Aldrich) was added to the
wells and incubated at room temperature until visible color was
observed (<30 min). Adding 50 .mu.Lof 1M H.sub.2SO.sub.4 stopped
the reaction and the plate was read at 495 nm (FlexStation 3
Reader, Molecular Devices, Sunnyvale, Calif., USA). For enzymatic
digestion, chondroitinase ABC (2 U/ml, Sigma) or hyaluronidase (500
IU/ml) was added to the plate for 3 hours at 37.degree. C. The
plate was then washed with PBST three times before incubation with
phage.
[0296] Results
[0297] To identify the potential protein targets of CAQK in the
brain tissue, mass spectrometry proteomics analysis was performed
of proteins separated from extracts of injured brain by affinity
chromatography on immobilized peptides. Table 1 shows a comparison
of proteins in eluates from CAQK and control (CGGK) columns. Among
the large number of proteins identified, peptides prominent in the
CAQK column eluates belonged primarily to the lectican family of
chondroitin sulfate proteoglycans (CSPGs (Ruoslahti, Glycobiology
6:489-492 (1996))). These included versican, associated proteins
tenascin-R and the hyaluronan and proteoglycan link protein
(Hapln). Versican and Hapln4 were exclusively present in the CAQK
column eluates. In normal brain, lectican sulfate proteoglycans
form extracellular matrix (ECM) complexes known as perineuronal
nets around neuronal surfaces (PNN) (Kwok et al., Int. J. Biochem.
Cell Biol. 44:582-586 (2012)), and the expression of some of these
lectican proteoglycans is upregulated at sites of CNS injury (Asher
et al., J. Neuroscience 22:2225-2236 (2002); Lau et al., Nat. Rev.
Neuroscience 14:722-729 (2013)).
TABLE-US-00001 TABLE 1 CAQK Binds to Brain ECM Proteins. Log.sub.2
LFQ intensity UniProt Gene CGGK CAQK Protein Name ID Name column
column Versican core Q62059 VCAN -- 25.98 protein Hyaluronan Q80WM4
HAPLN4 -- 19.84 and proteoglycan link protein 4 Tenascin-R Q8BYI9
TNR 24.14 26.71
[0298] The increase in expression of ECM-associated CSPGs at sites
of brain injury was confirmed by immunostaining. Versican,
tenascin-R, and the hyaluronan and proteoglycan link protein
(Hapln4), all of which are components of the brain ECM complex
upregulated following an injury, showed high expression in the
injured but not the uninjured hemisphere of the brain (FIG. 2). The
signal from intravenously injected CAQK co-localized with versican,
tenascin-R and Hapln4. The peptide signal also co-stained with WFA
(Wisteria floribunda agglutinin) lectin, a marker for PNNs. At the
cellular level, FAM-CAQK prominently accumulated at mature
oligodendrocytes identified by expression of the APC (Adenomatous
polyposis coli) marker. In several instances, the CAQK binding
pattern followed elongated cells that aligned in the direction of
the callosal axons. Only a few isolated Olig-2 and NG2-positve
cells, most likely oligodendrocyte progenitor cells, bound the
peptide. No CAQK was detected in or around other glial cell
populations, including astrocytes (GFAP+) and microglia.
Collectively, these data suggest that the binding molecule
(receptor) for CAQK peptide is present in the perineuronal net
(PNN) complex that is upregulated in brain injuries.
[0299] To explore further the association of CAQK with the PNN
complex, in vitro binding of CAQK phage to ECM produced by U251
human astrocytoma cells was tested. These cells express high levels
of versican and other members of the brain ECM (Dours-Zimmermann et
al., J. Biol. Chem. 269:32992-32998 (1994)), which suggests that
these cells are activated in culture. CAQK phage showed
significantly higher binding to the U251 ECM than a control phage
(FIG. 3A). In addition to providing further evidence for the ECM
binding of CAQK, this result indicates that CAQK recognizes the
human target. This is not surprising, as peptides are generally not
species-specific in their binding properties (Ruoslahti, Advanced
Mat. n/a-n/a (2012)). Binding to this ECM was specific as it was
inhibited with excess free CAQK peptide. Moreover, enzymatic
treatment of the ECM with chondroitinase ABC or hyaluronidase
resulted in loss of versican staining (FIG. 3D) and correspondingly
reduced CAQK binding. This suggests that the epitope for CAQK
resides in the PNN complex formed by the CSPGs, hyaluronic acid and
associated proteins (FIGS. 3B, 3C).
Example 4
CAQK as a Carrier of Diagnostics and Therapeutics to Brain
Injuries.
[0300] Materials and Methods
[0301] Synthesis and Functionalization of Porous Silicon
Nanoparticles (PSiNPs).
[0302] Single-crystalline highly doped p-type silicon wafers
(.about.1 m.OMEGA. cm resistivity, <100> polished,
boron-doped) were purchased from Virginia Semiconductor, Inc.
Porous silicon nanoparticles (PSiNPs) were prepared by
electrochemical perforation etching of the silicon wafers, as
described previously (Qin et al., Part. Part. Syst. Char.
31:252-256 (2014)). Briefly, the silicon wafer was anodically
etched in an electrolyte consisting of 3:1 (by volume) of 48%
aqueous HF:ethanol. Etching was carried out in a Teflon etch cell
that exposed the polished silicon wafer surface, using a platinum
coil counter electrode. The silicon wafer was contacted on the
backside with a strip of aluminum foil. The etching waveform
consisted of a square wave in which a lower current density of 50
mA/cm.sup.2 was applied for 1.8 sec, followed by a higher current
density of 400 mA/cm.sup.2 for 0.36 sec. This waveform was repeated
for 140 cycles, generating a perforated porous silicon film with
alternating layers of high and low porosity. The resulting porous
nanostructure was removed from the silicon substrate by applying a
current density of 3.7 mA/cm.sup.2 for 250 sec in an electrolyte
consisting of 1:30 (by volume) of 48% aqueous HF:ethanol. The
freestanding porous silicon films were then fractured to the
desired size (nominally 150 nm) by ultrasonication, and the
resulting nanoparticles were oxidized by immersion in aqueous borax
solution to activate photoluminescence (Joo et al., Adv. Funct.
Mat. 24:5688-5694 (2014)).
[0303] Characterization of PSiNPs. Transmission electron microscope
(TEM) images were obtained on JEOL-1200 EX II operating at 120 kV.
Dynamic light scattering (DLS, Zetasizer ZS 90, Malvern
Instruments) was used to determine the hydrodynamic size and Zeta
potential of the nanoparticles. Photoluminescence and fluorescence
spectra were obtained using a QE pro spectrometer (Ocean Optics).
Concentration of siRNA was determined by measuring absorbance at
260 nm using a spectrometer (NanoDrop 2000, Thermo Fisher
Scientific) based on the OD.sub.260 standard curve of siRNA.
[0304] Peptide Conjugation and siGFP Loading to PSiNPs. An aliquot
of PSiNPs (2 mg/ml in ethanol) was mixed with 20 .mu.L of
3-(ethoxydimethyl)-propylamine silane by vortex overnight at room
temperature. The amine-terminated nanoparticles were rinsed three
times with ethanol and then further reacted with 1 mL of
succinimidyl carboxy methyl ester-polyethylene glycol-maleimide (10
mg/ml in ethanol) for 2 hours, followed by rinsing with ethanol and
deionized water (three times each). An aqueous solution of the
peptides (500 .mu.L, 1 mg/ml, either CAQK or control) was added to
the resulting nanoparticles and mixed by vortexing for 2 hours to
conjugate the peptide via the free cysteine residue at the terminal
group of the peptide (Cai et al., Nat. Prot. 3:89-96 (2008)).
Peptide conjugation was confirmed by measuring fluorescence of a
FAM tagged to the peptide. The amount of peptide on nanoparticles
was estimated to be .about.70 nmol/mg (peptide/PSiNPs). The
oligonucleotide siGFP was electrostatically loaded to the
positively charged porous inner structure of the nanoparticles by
mixing with siRNA solution (200 .mu.M) at 4 .degree. C. for 24 hr.
The loading amount of siGFP was .about.7 wt % (siGFP/PSiNP), which
was determined by measuring absorbance at 260 nm. Note that the PEG
linkers were attached only onto the outer surface (not the inside
pores) of PSiNPs due to their large molecular weight and long chain
length relative to the pore size (.about.12 nm).
3-(ethoxydimethyl)-propylamine silane and succinimidyl carboxy
methyl ester-polyethylene glycol-maleimide (SCM-PEG-Mal, MW 5000)
were purchased from Sigma-Aldrich and Laysan Bio, respectively, and
used as received without further purification. RNase free water was
purchased from Thermo Fischer (Carlsbad, Calif.). Small interfering
RNA against green fluorescent protein (siGFP) was purchased from
Dharmacon. The sequences for the sense and antisense strands of
siGFP are: 5'-GGCUACGUCCAGGAGCGCACCdTdT-3' (sense) (SEQ ID NO:1)
and 5'-UGCGCUCCUGGACGUAGCCTTdTdT-3' (antisense) (SEQ ID NO:2).
[0305] In vivo siRNA Targeting and Analysis. Transgenic CAG-GFP
mice were purchased from The Jackson Laboratory (stock #006567).
Brain injuries were done as described above. Peptide-conjugated and
siRNA-loaded PSiNPs (300 .mu.g) were administered twice via
tail-vein injections at 6 and 24 hours post injury (n=3). Three
days after injury, mice were perfused, organs harvested and fixed
for downstream analysis. The tissues were imaged under a time-gated
imaging setup and the GFP expression was analyzed by researchers
blinded to the experimental groups.
[0306] Gated Luminescence Imaging of Silicon Nanoparticles
(GLISiN). Gated luminescence images were acquired from a
custom-built time-domain imaging system using an intensified CCD
camera (iSTAR 334T, Andor Technology Ltd.), as reported (Joo et
al., ACS Nano 9:6233-6241 (2015)). A tunable laser consisting of a
tripled Nd:YAG-pumped optical parametric oscillator (Opolette 355,
Opotek Inc.) were used as an excitation source at a repetition rate
of 10 Hz, which was synchronized and triggered with the CCD. The
Andor SOLIS software was used to control time delays and
acquisition conditions and to analyze signal-to-noise ratio (SNR).
Mouse tissues were placed on a black polystyrene plate, and bright
field (under ambient light) and gated luminescence (under
excitation with pulsed laser, .lamda..sub.ex=410 nm) images were
taken.
[0307] Human Tissue Experiments. Formalin fixed human brain tissues
were obtained from the Brain Tissue Repository maintained by the
Center for Neuroscience & Regenerative Medicine (CNRM) at the
Uniformed Services University of the Health Sciences (USU) in
Bethesda, Md. The TBI case is from a patient with moderate TBI
(automobile accident) who died at age 72. The control case is from
a 63 year-old male without any neurologic diagnosis or any signs of
TBI on detailed neuropathologic evaluation. Fixed tissues were
cryopreserved and sectioned for overlay binding with AgNPs as
described above. For immunohistochemistry, an antigen retrieval
step was done prior to incubation with anti-Hapln4 and
anti-versican antibodies.
[0308] Statistical Analysis. All data represent mean value .+-.SEM.
All the significance analysis was done using Statistica 8.0
software, using one-way ANOVA or two-tailed heteroscedastic
Student's t test. The details of the statistical tests carried out
are indicated in respective figure legends.
[0309] Results
[0310] The accumulation of the FAM label attached to the CAQK
peptide suggested that CAQK is capable of delivering low molecular
weight compounds into sites of brain injury. To investigate further
the translational potential of the CAQK targeting approach,
CAQK-mediated delivery to brain injury of nanoparticles (NPs) was
first examined as a model of both an imaging agent and a drug
carrier. CAQK-conjugated, silver NPs (mean diameter--20 nm),
administered intravenously, showed significantly greater
accumulation in injured brain tissue than control NPs (FIG. 4A).
The localization of CAQK-NPs was in excellent agreement with the
localization of the free CAQK peptide. Thus, CAQK targeted NPs
mimic the homing ability of the free peptide.
[0311] To demonstrate the versatility of the CAQK system, delivery
of oligonucleotides loaded into porous silicon NPs as a carrier was
tested (Park et al., Nat. Mater. 331-336 (2009)). The proof of
concept approach was to silence local expression of green
fluorescent protein (GFP) systemically expressed in transgenic mice
from the CAG promoter (Okabe et al., FEBS Lett 407:313-319 (1997)).
Therapeutic oligonucleotides were simulated by using siRNA against
GFP loaded in CAQK conjugated porous silicon nanoparticles
(CAQK-PSiNP) (FIGS. 4B-4D). PSiNPs were intravenously injected into
the GFP mice with PBI and visualized by time-gated luminescence
imaging (Joo et al., ACS Nano 9:6233-6241 (2015); Gu et al., Nat.
Commun. 4:2326 (2013)), allowing quantification of their
accumulation in the excised brains. The imaging showed that
CAQK-PSiNPs accumulated in the injuries at markedly higher (35
fold) levels than PSiNPs coated with a control peptide. Other
tissues, including regions of the brain outside the injury area,
showed no significant difference in the accumulation of CAQK and
control PSiNPs (FIG. 5B). Confocal microscopy on transverse
cortical sections from mice injected with CAQK-PSiNP-siGFP
exhibited a large void of GFP expression at the injury site,
whereas brains from mice treated with control NPs did not differ
from untreated brains. A 70% silencing of GFP expression was
observed by targeting siGFP, whereas minimal silencing was observed
with untargeted siGFP and other controls. This silencing was
visible across the entire injury and not just in a particular cell
type due to the gradual degradation of PSiNP and release of the
siGFP over time in the injury. The gene silencing was specific for
brain injury, as GFP expression remained unaltered in normal brains
or in other major tissues.
[0312] Lastly, to examine CAQK recognition of human brain injury,
ex vivo binding of CAQK-conjugated silver NPs was tested on human
cortical sections obtained from a head trauma patient. The CAQK-NPs
showed intense binding to the injured brain sections from the
cortex and the corpus callosum areas, whereas binding to normal
brain sections was minimal (FIGS. 6A and 6B). Similar to the mouse
brains, a significant elevation in expression of versican and
Hapln4 was observed in injured brain compared to normal brain by
immuno-histochemistry (FIGS. 6C-6E). These findings confirm CAQK
binding to human target and support its potential utility for
therapeutic application in humans.
[0313] Discussion
[0314] Two mouse models were used in this study. The penetrating
brain injury model mimics gunshot or shrapnel wounds, such as the
ones sustained by a warfighter. The blunt cortical impact model
more generally reproduces the features of severe TBI. CAQK
recognized the injuries in both models, suggesting broad utility
across acute brain injuries. The fact that the contralateral
hemisphere, unlike normal brain, accumulated some CAQK phage
indicating that less severe injuries than the ones used here can
also be targeted with CAQK. CAQK will also home to spinal cord
injuries and demyelinating CNS lesions such as in multiple
sclerosis to the extent they express the target of CAQK. CAQK
recognition of its target in cultured human cells and in cortical
sections of injured human brain has been demonstrated.
[0315] The phage screening in this study revealed a novel aspect of
the in vivo screening: whereas the previous screens have probed the
vasculature of the target tissue, even in the brain (Chen et al.,
Nat. Med. 15:1215-1218 (2009); Pasqualini et al., Nature
380:364-366 (1996); Fan et al., Pharm. Res. 24:868-879 (2007)), the
compromised BBB integrity in brain injury allowed the phage to
probe the extravascular brain tissue. Secondly, the high-throughput
sequencing of the peptide-encoding inserts in the phage genome
provided a technical improvement to the screening. One round of
selection, as opposed to repeated rounds as previously done,
provided a fingerprint of over 200,000 peptide sequences, revealing
a striking enrichment of phage displaying the CAQK peptide
sequence. Although a cyclic phage library was used, a library of
this design contains a minority of linear peptides because stop
codons occur within the random insert causing truncation of the
cyclic peptide. Additionally, mutations may also change the
structure of the peptide. Thus, peptides that do not conform to the
general structure of the library are commonly encountered in phage
screening (Hoffman et al., Cancer Cell 4:383-391 (2003); Simberg et
al., PNAS 104:932-936 (2007)). The recovery of cyclic peptides
containing the CAQK motif, in addition to the dominant linear CAQK
peptide, suggests that CAQK motif is also active in the context of
a cyclic peptide.
[0316] BBB disruption is an important contributor to secondary
injury following TBI, and therapies to restore BBB functionality
are under investigation for neuroprotection (Pillai et al., J.
Cerebral Flow and Metabol. 29:1846-1855 (2009)). The localized
permeability of BBB and the delayed onset of secondary injury
provide a window of opportunity for therapeutic intervention. The
literature (Cunningham et al., J. Neurotrauma 31:505-514 (2014))
and the results shown here suggest the duration of the BBB
impairment is at least up to 5 days. Within this time window,
affinity ligand-based (synaphic) targeting can be an effective drug
delivery approach; results show as high as 35-fold enhancement in
the accumulation of systemically administered imaging agents and
therapeutics at and around the site of injury.
[0317] The concentrating effect of synaphic targeting is likely
accounted for by two factors: the peptide can access and bind to
its target that allows accumulation of the payload and causes
retention at the site of injury. The impairment of the BBB allows
all circulating substances to enter the injury area. And, if the
peptide receptor is sufficiently abundant relative to the amount of
the peptide-drug conjugate used, the binding of the peptide to the
receptor can drive payload accumulation beyond what is caused by
passive leakage (Hussain et al., Scien. Rep. 4:5232 (2014)). This
is the case in brain injury, where the components of the CSPG
complex are overexpressed upon an injury (Kwok et al., Int. J.
Biochem. Cell Biol. 44:582-586 (2012)) (FIG. 2). The second
important factor is the retention effect. As the drug concentration
decreases in circulation, the drug is washed out of the injury area
(Stewart et al., J. Neurosci. Meth. 41:75-84 (1992)). Peptide
binding to its target can retain the drug in the injured
microenvironment by minimizing this washout. Therefore, the
targeting approach in this work encompasses the critical period of
healing, which may provide a more lasting therapeutic effect, at
least when the therapeutic action is long-acting. Notably, some
oligonucleotides, which is one of the types of drugs that were
successfully delivered in this work, have been shown to remain
active for weeks in tissues (Bartlett et al., Nuc. Acids Res.
34:322-333 (2006)).
[0318] CNS injury results in formation of a CSPG-rich glial scar,
which is a major barrier to regeneration (Silver et al., Nat. Rev.
Neurosci. 5:146-156 (2004)). Strategies to prevent the accumulation
of CSPGs in injury or dissolve existing deposits have been explored
(Lau et al., Nat. Rev. Neurosci. 14:722-729 (2013)). However,
site-specific delivery of the active compounds has been a
challenge. The intrinsic affinity of CAQK peptide for CSPG rich
areas in injured brain could be effective in directing a
CSPG-reducing payload, such as the chondroitinase ABC enzyme (Hill
et al., PNAS 109:9155-9160 (2012)). Having successfully targeted
nanoparticle payload into brain injuries indicates that the same
can be accomplished with proteins, such as chondroitinase. The
ability of the present approach to concentrate a payload at the
site of brain injury is useful for reducing toxicity at off-target
sites. An example of an existing therapeutic agent that would
benefit from reduced toxicity is the neuro-protective agent Brain
Derived Neurotrophic Factor (BDNF) (Nagahara et al., Nat. Rev. Drug
Disc. 10:209-219 (2011)). It has side effects in the normal brain,
which CAQK does not target. Thus, the targeting approach disclosed
here has the potential of converting agents with unfavorable
pharmacokinetic profile into efficient drugs.
[0319] Oligonucleotide-based drugs are a new class of drugs with
great potential but hampered by delivery problems in vivo. An
example is siRNA, a therapeutic modality with the desired
characteristics of specificity and potency, but particularly
difficult to deliver through systemic circulation. Previous studies
on siRNA therapy of brain injuries have either used direct
injection into the CNS space or silenced a target present in the
brain endothelial cells (Fukuda et al., Genes 4:435-456 (2013)).
The CAQK-mediated targeted delivery of siRNA reported here provides
the first evidence of delivery of active siRNA into injured brain
tissue from systemic circulation. A number of targets for gene
silencing (such as Bcl-2 family proteins, caspases, HDAC, and PTEN)
have been suggested for brain injury (Fukuda et al., Genes
4:435-456 (2013)). Here, CAQK-mediated siRNA delivery was
accomplished by using porous nanoparticles as a carrier. Thus,
these results also open up brain injuries for nanomedicine-based
therapeutic approaches.
[0320] The discovery of CAQK is therapeutically useful because it
can direct a payload from systemic circulation to the site of acute
brain injury and retain it there for therapeutically relevant
timescales. This approach provides an alternative to local
delivery, which is invasive and can add complications to the
injury. Moreover, the approach is applicable to human patients
because CAQK recognizes the human target molecule and because the
expression of the target appears to be elevated in injured human
brain tissues in the same way it is in the mouse injuries.
[0321] It is understood that the disclosed method and compositions
are not limited to the particular methodology, protocols, and
reagents described as these may vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended
claims.
[0322] It must be noted that as used herein and in the appended
claims, the singular forms "a ", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a cargo molecule" includes a plurality of
such cargo molecules, reference to "the cargo molecule" is a
reference to one or more cargo molecules and equivalents thereof
known to those skilled in the art, and so forth.
[0323] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises," means "including but not limited to,"
and is not intended to exclude, for example, other additives,
components, integers or steps.
[0324] "Optional" or "optionally" means that the subsequently
described event, circumstance, or material may or may not occur or
be present, and that the description includes instances where the
event, circumstance, or material occurs or is present and instances
where it does not occur or is not present.
[0325] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, also specifically contemplated and
considered disclosed is the range from the one particular value
and/or to the other particular value unless the context
specifically indicates otherwise. Similarly, when values are
expressed as approximations, by use of the antecedent "about," it
will be understood that the particular value forms another,
specifically contemplated embodiment that should be considered
disclosed unless the context specifically indicates otherwise. It
will be further understood that the endpoints of each of the ranges
are significant both in relation to the other endpoint, and
independently of the other endpoint unless the context specifically
indicates otherwise. It should be understood that all of the
individual values and sub-ranges of values contained within an
explicitly disclosed range are also specifically contemplated and
should be considered disclosed unless the context specifically
indicates otherwise. Finally, it should be understood that all
ranges refer both to the recited range as a range and as a
collection of individual numbers from and including the first
endpoint to and including the second endpoint. In the latter case,
it should be understood that any of the individual numbers can be
selected as one form of the quantity, value, or feature to which
the range refers. In this way, a range describes a set of numbers
or values from and including the first endpoint to and including
the second endpoint from which a single member of the set (i.e. a
single number) can be selected as the quantity, value, or feature
to which the range refers. The foregoing applies regardless of
whether in particular cases some or all of these embodiments are
explicitly disclosed.
[0326] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed method and compositions
belong. Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present method and compositions, the particularly useful
methods, devices, and materials are as described. Publications
cited herein and the material for which they are cited are hereby
specifically incorporated by reference. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such disclosure by virtue of prior invention.
No admission is made that any reference constitutes prior art. The
discussion of references states what their authors assert, and
applicants reserve the right to challenge the accuracy and
pertinency of the cited documents. It will be clearly understood
that, although a number of publications are referred to herein,
such reference does not constitute an admission that any of these
documents forms part of the common general knowledge in the
art.
[0327] Although the description of materials, compositions,
components, steps, techniques, etc. may include numerous options
and alternatives, this should not be construed as, and is not an
admission that, such options and alternatives are equivalent to
each other or, in particular, are obvious alternatives. Thus, for
example, a list of different cargo molecules does not indicate that
the listed cargo molecules are obvious one to the other, nor is it
an admission of equivalence or obviousness.
[0328] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the method and
compositions described herein. Such equivalents are intended to be
encompassed by the following claims.
Sequence CWU 1
1
9123DNAArtificial SequencesiRNA for sense strand of
siGFPmisc_feature(22)..(23)deoxythymidine 1ggcuacgucc aggagcgcac
ctt 23223DNAArtificial SequencesiRNA for antisense strand of
siGFPmisc_feature(22)..(23)deoxythymidine 2ugcgcuccug gacguagcct
ttt 2339PRTArtificial SequenceSynthetic
PeptideMISC_FEATURE(2)..(8)Xaa = any amino acid 3Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys 1 5 44PRTArtificial SequenceSynthetic Peptide
4Cys Ala Gln Lys 1 54PRTArtificial SequenceSynthetic
PeptideMISC_FEATURE(1)..(1)N-terminal FAM tag 5Cys Ala Gln Lys 1
64PRTArtificial SequenceSynthetic Peptide 6Cys Gly Gly Lys 1
74PRTArtificial SequenceSynthetic
PeptideMISC_FEATURE(1)..(1)N-terminal FAM tag 7Cys Gly Gly Lys 1
86PRTArtificial SequenceSynthetic
PeptideMISC_FEATURE(1)..(1)N-terminal acetyl
modificationMISC_FEATURE(6)..(6)C-terminal amide modification 8Cys
Cys Pro Gly Cys Cys 1 5 95PRTArtificial SequenceSynthetic
PeptideMISC_FEATURE(1)..(1)N-terminal FAM
tagMISC_FEATURE(1)..(1)Xaa = any amino
acidMISC_FEATURE(5)..(5)C-terminal NH2 modification 9Xaa Cys Ala
Gln Lys 1 5
* * * * *